Oligonucleotide compositions and methods for treating disease including inflammatory conditions

ABSTRACT

The invention relates to therapeutic antisense oligonucleotides directed against genes coding for phosphodiesterase (PDEs) and the use of these in combination. These antisense oligonucleotides may be used as analytical tools and/or as therapeutic agents in the treatment of disease associated with reduced cellular cAMP in a patient, such as inflammatory diseases of the respiratory tract including, for example, asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome, bronchitis, chronic bronchitis, silicosis, pulmonary fibrosis, lung allograft rejection, allergic rhinitis and chronic sinusitis as well as other conditions in which an increase in cyclic AMP or a decrease in PDE levels is beneficial.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 10/953,512filed on Sep. 29, 2004, which claims the benefit under 35 U.S.C § 119(e)of U.S. provisional Application No. 60/507,016, filed Sep. 29, 2003, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods, reagents and compositions of use forantisense oligonucleotide-based therapy. In particular, the inventionrelates the application of antisense oligonucleotide-based therapy inthe treatment of disease associated with reduced cAMP in a patientincluding, for example, PDE-related disease such as inflammatoryconditions. The invention also relates to gene therapy methods andmethods for identifying novel antisense-based strategy wherein cyclicAMP phosphodiesterases are involved.

BACKGROUND OF THE INVENTION

The alveolar and airway epithelium is recognized as a dynamic barrierthat plays an important role in regulating the inflammatory andmetabolic responses to oxidative stress, sepsis, endotoxemia, and othercritical illnesses in the lung. The respiratory epithelium, inparticular, is a primary target of an inflammatory/infectious conditionat the epithelial-blood interface, and is itself capable of amplifyingan inflammatory signal by recruiting inflammatory cells and producinginflammatory mediators.

Chronic Obstructive Pulmonary Disease (COPD) is one example of aninflammatory airway and alveolar disease where persistent upregulationof inflammation is thought to play a role. Inflammation in COPD ischaracterized by increased infiltration of neutrophils, CD8 positivelymphocytes, and macrophages into the airways. Neutrophils andmacrophages play an important role in the pathogenesis of airwayinflammation in COPD because of their ability to release a number ofmediators including elastase, metalloproteases, and oxygen radicals thatpromote tissue inflammation and damage. It has been suggested thatinflammatory cell accumulation in the airways of patients with COPD isdriven by increased release of pro-inflammatory cytokines and ofchemokines that attract the inflammatory cells into the airways,activate them and maintain their presence. The cells that are presentalso release enzymes (like metalloproteases) and oxygen radicals whichhave a negative effect on tissue and perpetuate the disease. A vastarray of pro-inflammatory cytokines and chemokines have been shown to beincreased within the lungs of patients with COPD. Among them, animportant role is played by tumor necrosis factor alpha (TNF-alpha),granulocyte-macrophage colony stimulating factor (GM-CSF) andinterleukin 8 (IL-8), which are increased in the airways of patientswith COPD.

Other examples of respiratory diseases where inflammation seems to playa role include: asthma, eosinophilic cough, bronchitis, acute andchronic rejection of lung allograft, sarcoidosis, pulmonary fibrosis,rhinitis and sinusitis. Asthma is defined by airway inflammation,reversible obstruction and airway hyperresponsiveness. In this diseasethe inflammatory cells that are involved are predominantly eosinophils,T lymphocytes and mast cells, although neutrophils and macrophages mayalso be important. A vast array of cytokines and chemokines have beenshown to be increased in the airways and play a role in thepathophysiology of this disease by promoting inflammation, obstructionand hyperresponsiveness.

Eosinophilic cough is characterized by chronic cough and the presence ofinflammatory cells, mostly eosinophils, within the airways of patientsin the absence of airway obstruction or hyperresponsiveness. Severalcytokines and chemokines are increased in this disease, although theyare mostly eosinophil directed. Eosinophils are recruited and activatedwithin the airways and potentially release enzymes and oxygen radicalsthat play a role in the perpetuation of inflammation and cough.

Acute bronchitis is an acute disease that occurs during an infection orirritating event for example by pollution, dust, gas or chemicals, ofthe lower airways. Chronic bronchitis is defined by the presence ofcough and phlegm production on most days for at least 3 months of theyear, for 2 years. One can also find during acute or chronic bronchitiswithin the airways inflammatory cells, mostly neutrophils, with a broadarray of chemokines and cytokines. These mediators are thought to play arole in the inflammation, symptoms and mucus production that occurduring these diseases.

Lung transplantation is performed in patients with end stage lungdisease. Acute and more importantly chronic allograft rejection occurwhen the inflammatory cells of our body, lymphocytes, do not recognizethe donor organ as “self”. Inflammatory cells are recruited bychemokines and cytokines and release a vast array of enzymes that leadto tissue destruction and in the case of chronic rejection a diseasecalled bronchiolitis obliterans.

Sarcoidosis is a disease of unknown cause where chronic non-caseatinggranulomas occur within tissue. The lung is the organ most commonlyaffected. Lung bronchoalveolar lavage shows an increase in mostlylymphocytes, macrophages and sometimes neutrophils and eosinophils.These cells are also recruited and activated by cytokines and chemokinesand are thought to be involved in the pathogenesis of the disease.

Pulmonary fibrosis is a disease of lung tissue characterized byprogressive and chronic fibrosis (scarring) which will lead to chronicrespiratory insufficiency. Different types and causes of pulmonaryfibrosis exist but all are characterized by inflammatory cell influx andpersistence, activation and proliferation of fibroblasts with collagendeposition in lung tissue. These events seem related to the release ofcytokines and chemokines within lung tissue.

Acute rhinitis is an acute disease that occurs during an infection orirritating event, for example, by pollution, dust, gas or chemicals, ofthe nose or upper airways. Chronic rhinitis is defined by the presenceof a constant chronic runny nose, nasal congestion, sneezing andpruritis. One can also find within the upper airways during acute orchronic rhinitis inflammatory cells with a broad array of chemokines andcytokines. These mediators are thought to play a role in theinflammation, symptoms and mucus production that occur during thesediseases.

Acute sinusitis is an acute, usually infectious disease of the sinusescharacterized by nasal congestion, runny, purulent phlegm, headache orsinus pain, with or without fever. Chronic sinusitis is defined by thepersistence for more than 6 months of the symptoms of acute sinusitis.One can also find during acute or chronic sinusitis within the upperairways and sinuses inflammatory cells with a broad array of chemokinesand cytokines. These mediators are thought to play a role in theinflammation, symptoms and phlegm production that occur during thesediseases.

As described above, these inflammatory respiratory diseases are allcharacterized by the presence of mediators that recruit and activatedifferent inflammatory cells which release enzymes or oxygen radicalscausing symptoms, the persistence of inflammation and when chronic,destruction or disruption of normal tissue.

A logical therapeutic approach would be to downregulate cytokine andchemokine production and the inflammatory cell response. This has beenperformed in all the diseases described above by employing eithertopical or systemic corticosteroids with different levels of success.Corticosteroids are immune suppressive and have effects not only oninflammatory cells but also on other cells of the body that lead totoxicity when administered chronically.

Despite the availability of medications for COPD, asthma and otherinflammatory respiratory diseases, the prevalence and morbidity of thesediseases has remained stable or increased. It is obvious that there isan unmet medical need for the therapy of inflammatory respiratorydiseases, and innovative therapeutic agents are urgently required.Antisense oligonucleotide-based therapy offers a new alternativeapproach to selectively decrease the expression of specific geneswithout the undesirable toxic effects of traditional therapeuticstrategies. Antisense therapies are being investigated for the treatmentof several diseases. It has been previously shown that antisenseoligonucleotides directed against receptors for inflammatory mediatorscan be administered to the lungs and down-regulate their targets asdescribed in WO9966037.

A therapeutic approach that would decrease pro-inflammatory cytokine andchemokine release by a vast array of cells while having a reduced effecton the release of anti-inflammatory mediators or enzymes may have anadvantage over current therapies for inflammatory respiratory diseasesor any other systemic inflammatory disease.

The cyclic nucleotides cAMP and cGMP are ubiquitous second messengersparticipating in signaling transduction pathways and mechanisms.Mammalian cells have evolved a complex and highly conserved complementof enzymes that regulate the generation and inactivation of cyclicnucleotides through multiple and complex feedforward and feedbackmechanisms. Both cAMP and cGMP are formed from their respectivetriphosphates (ATP and GTP) by the catalytic activity of adenylyl(adenylate) or guanylyl (guanylate) cyclase, respectively as describedin Essayan D. M. Cyclic nucleotide phosphodiesterase (PDE) inhibitorsand immunomodulation. Biochem. Pharmacol., 1999, 57, 965-973.Inactivation of cAMP/cGMP is achieved by hydrolytic cleavage of the3′-phosphodiester bond catalyzed by the cyclic-nucleotide-dependentphosphodiesterases (PDEs), resulting in the formation of thecorresponding, inactive 5′-monophosphate as described in Essayan, 1999and Perry M. J. and Higgs G. A. Chemotherapeutic potential ofphosphodiesterase inhibitors. 1998, Curr Opin Chem Biol. 4:472-81.

It has been shown that the inflammatory response and its progression isexquisitely sensitive to modulations in the steady-state levels ofcyclic nucleotides, where target cells for their effects extend beyondimmune cells to include accessory cells, such as airway smooth muscle,epithelial and endothelial cells, and neurons as described in Perry andHiggs, 1998; Essayan, 1999. In this respect, the emerging concept thatmodulation of intracellular cyclic nucleotides plays a major role inregulating the inflammatory milieu has recently evolved into targetingand improving inflammatory/autoimmune responses. The cyclic nucleotidePDEs are a large, growing multigene family, comprising at least 11families of PDE enzymes. The profile of selective and nonselective PDEinhibitors in vitro and in vivo, therefore, suggests a potentialtherapeutic utility as antidepressants, antiproliferative,immunomodulatory, tocolytics, inotropes/chronotropes, and cytoprotectiveagents.

Intracellular cAMP seems to have a fundamental role, not only in smoothmuscle relaxation, activation and proliferation but also in themodulation of the release of mediators by inflammatory cells. DecreasedcAMP levels can lead to increased production of inflammatory mediatorssuch as TNF-alpha, GM-CSF, and IL-8 in airway epithelial cells.

Insight into the molecular mechanisms of the regulatory role ofcytokines in cellular homeostasis as well asinflammatory/autoimmune/infectious diseases has begun to provide newapproaches to design therapeutic strategies for pharmacologicalinterventions. One such novel approach is the chemotherapeutic potentialof PDE enzyme blockade, which revealed a phenomenal diversity andcomplexity scheme for promising therapeutics across a broad spectrum ofdisease states. One mechanistic understanding of PDE inhibition iscentered on the immunomodulatory properties of cyclic nucleotides(cAMP/cGMP), thereby paving a channel through which anti-inflammatory,therapeutic applications could be clearly demonstrated.

The inflammatory response and its progression is exquisitely sensitiveto modulations in the steady-state levels of cyclic nucleotides, wheretarget cells for their effects extend beyond immune cells to includestructural cells, such as epithelial, smooth muscle and endothelialcells, and neurons (Perry and Higgs, 1998; Essayan, 1999). Modulation ofintracellular cyclic nucleotides plays a major role in regulating theinflammatory response. The cyclic nucleotide PDEs are a large, growingmultigene family, comprising at least 11 families of PDE isoenzymes.PDEs differ in their tissue and cellular distribution as well as intheir molecular and physicochemical characteristics includingnucleotides and protein sequences, substrate specificity, inhibitorsensitivity, and cofactor requirements. Within different families,tissue specific isoforms are generated from the same gene by alternativemRNA splicing and differential promoter usage.

The cAMP-specific PDE4 enzyme family is one of the most extensivelystudied PDEs. Enzymes within this family are found in mostpro-inflammatory and immune cells, where they play a key role in theregulation of cAMP metabolism. PDE4 enzymes are expressed inmacrophages, neutrophils, cytotoxic CD8+ T cells, bronchial epithelialcells, and airway smooth muscle cells. Moreover, PDE4 inhibitorsmodulate inflammation in animal models of respiratory diseases,suggesting that PDE4 may represent a suitable target in a therapeuticbased strategy for intervention with small molecule inhibitors.Anti-PDE4 drugs inhibit the hydrolysis of intracellular cAMP, which inturn provides bronchodilation and suppression of the inflammatoryresponse. Selective PDE4 inhibitors such as cilomilast and roflumilastare active in animal models of neutrophil inflammation (Bundschuch D Set al. J. Pharmacol. Exp. Ther. 2001, 297: 280-290). Although the use ofPDE4 inhibitors for the treatment of airway inflammation is underintensive clinical investigations, several inhibitors have been droppedbecause of their toxicity, dose-limiting side effects, of which nauseaand vomiting are the most common physiological manifestations.Consequently, improving the therapeutic ratio of PDE4 inhibitors raiseda major challenge that is still an important field of investigation.

Considering the distribution of enzymes in target tissues, with highactivity of PDE3 and PDE4 in airway smooth muscle and inflammatorycells, selective inhibitors of these enzymes may add to the therapy ofchronic airflow obstruction. Generally small molecule inhibitors havebeen focused on one or several PDE without assessing the potentialdetrimental effects of inhibiting all the isoenzymes of a PDE. Forexample it has been suggested that most of the toxicity attributed tothe PDE4 antagonists will occur through inhibition of the PDE4 isotypeD. In addition, as shown herein, inhibition of certain isoenzymes ofPDEs does not decrease all pro-inflammatory mediators however, acombination of isotype specific oligonucleotides can lead to an effectthat is much broader when the right combination of antisenseoligonucleotides is employed.

The PDE3 family contains two different genes, PDE3A and PDE3B, which arecGMP-inhibited and display a high affinity towards cAMP. Each PDE3 genecodes for at least two splice isoforms. The PDE3A has been identified insmooth muscle, platelets and cardiac tissues. The PDE3B is most abundantin adipocytes and liver cells. However, initial data from clinicaltrials with selective PDE3 inhibitors or a combination with PDE4inhibitors have been somewhat disappointing and have tempered theexpectations considerably since these drugs had limited efficacy andtheir use was clinically limited through side effects.

PDE7 was first isolated from a human glioblastoma. PDE7A codes for acAMP-specific PDE that is insensitive to cGMP and inhibitors of PDE3 andPDE4 and has an amino acid sequence distinct from other cAMP PDEs. Inhumans two genes (PDE7A and PDE7B) have been characterized. The PDE7Agene codes for three isoenzymes (PDE7A1, PDE7A2, and PDE7A3) derivedfrom the same gene by alternative mRNA splicing. In humans, PDE7A2 mRNAis expressed abundantly in skeletal muscle, heart, and kidney, whereasthe testis, lung, and immune system (thymus, spleen, lymph node, bloodleukocytes) are rich sources of PDE7A1. In addition, activated, but notnaïve, human T lymphocytes express the splice variant PDE7A3. Incontrast, PDE7B exists as a single isoenzyme in humans, it shares ˜70%sequence similarity to PDE7A, but distinct kinetic properties. PDE7B isexpressed predominantly in the brain and in a number of other tissuesincluding liver, heart, thyroid glands, and skeletal muscle. Stimulationof human naïve T cells with anti-CD3 and anti-CD28 antibodies has beenshown to promote IL-2 production and clonal amplification. These effectswere attributed to PDE7A and down regulation of this enzyme preventslymphocyte proliferation (Li L et al., Science, 1999, 283, 848-851).

The PDE4 family contains 4 different genes (PDE4 A-D). Due toalternative splicing of the genes, multiple splice variants are reportedand classified into two main groups, the long and the short forms.PDE4A, B and D gene products are found in most immune and inflammatorycells. These are present either constitutively or after activation asdescribed in Burnouf C. and Pruniaux M. P. Current Pharmaceutical Design2002; 8:1255-1296. Inhibition of all or certain isotypes of PDE4 isassociated with downregulation of several inflammatory mediators;however, an increase in certain pro-inflammatory cytokines (e.g. IL-6)has been described (Giembycz M. A. Expert Opin Invest Drugs 2001,10:1361-1379).

It would therefore be desirable to inhibit all three PDE enzymes;however this approach may be plagued by the toxicity that has beendescribed with administration of inhibitors of each of these enzymes.The topical application of antisense oligonucleotides could circumventthe systemic toxicity of enzyme inhibition but there appear to be toomany isoenzymes of these PDE for this approach to be practical.Inhibition of one or several isotypes of PDE enzymes may not be aseffective as full PDE inhibition since other isotypes would be presentto have their pro-inflammatory effects.

It would therefore appear desirable to seek a way to down-regulatepro-inflammatory mediators and their cells while affecting lessanti-inflammatory mediators or inhibitory enzymes for the therapy ofinflammatory respiratory diseases. The therapeutic application of acombination of antisense oligonucleotides directed against selectedisotypes of PDE enzymes is therefore herein proposed as a therapy forinflammatory respiratory diseases or any disease where increased cyclicAMP plays a role.

SUMMARY OF THE INVENTION

The present invention provides antisense oligonucleotide compounds thatare effective at down-regulating PDE isotype genes that they aredirected against as well as selected antisense oligonucleotide compoundsthat are effective at down-regulating not only the PDE isotype genesthey are directed against but also other related genes including otherPDE isotype genes and inflammatory genes. The present invention alsoprovides a composition comprising a combination of at least twoantisense oligonucleotide compounds each directed against a differentPDE target gene and each being effective to downregulate or inhibit aPDE target gene, each oligonucleotide compound being present in thecombination at a concentration at which it exhibits less than 20%inhibition of its target gene, the combination of the oligonucleotidecompounds exhibiting more than 20% inhibition and at least doubling theinhibition of at least one of the target genes. The present inventionfurther provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a combination of at least twoantisense oligonucleotides as described above.

The combinations of the present invention appear to exert an inhibitoryaction by an approach that has been termed multiple gene knock down.Multiple gene knock down encompasses situations in which:

1) an antisense oligonucleotide downregulates not only the gene that itis directed against but also downregulates other related genes; and2) a combination of at least 2 antisense oligonucleotides, each presentat a concentration at which the antisense oligonucleotide is practicallyineffective to downregulate the gene it is directed against on its own,the combination leading to significant downregulation of both genes thatthe antisense oligonucleotides are directed against and optionally otherrelated genes.

The present invention uses the above approach to provide methods,compositions and kits for treating and/or preventing disease associatedwith reduced cAMP in a patient, including PDE-related disease andinflammatory disease including respiratory diseases and moreparticularly COPD and asthma.

The present invention provides a composition comprising at least 2antisense oligonucleotide compounds, each antisense oligonucleotidecompound being capable of downregulating a different gene, eachantisense oligonucleotide compound being present at a concentration atwhich the antisense oligonucleotide compound is practically ineffectiveon its own to downregulate the gene it is directed against, thecombination of the at least 2 antisense oligonucleotide compoundsleading to a significant downregulation of each of the genes that theantisense oligonucleotide compounds are directed against and optionallyother related genes. The present invention also provides for the use ofthe composition in the treatment and/or prevention of inflammatoryrespiratory diseases.

The present invention provides a composition comprising at least 2antisense oligonucleotides capable of downregulating different PDEisozyme genes, each oligonucleotide being present at a concentration atwhich the oligonucleotide is practically ineffective on its own todownregulate the PDE gene it is directed against, the combination of theat least 2 oligonucleotides leading to a significant downregulation ofboth genes that the oligonucleotides are directed against and optionallyother related genes. The present invention also provides for the use ofthe composition in the treatment and/or prevention of inflammatoryrespiratory diseases.

The invention also provides methods, reagents and compositions forreducing the expression and consequently the activity of cell cyclic-AMPphosphodiesterases and thereby maintaining the correct balance ofintracellular c-AMP and cytokine/chemokine production.

The invention further provides methods and tools foridentifying/screening in vitro, in vivo and ex vivo, combinations ofnovel antisense oligonucleotide compounds, drugs and vaccines that arecapable of interfering with PDE expression and elevating theintracellular c-AMP level.

The present invention also provides gene-based therapy and transfectionmethods in which one or more antisense oligonucleotide compounds areused for cell transfection or delivered to humans and animals forinterfering with PDE isotype gene expression.

The present invention further provides antisense oligonucleotideseffective against PDE3 isotypes, such as PDE3A and PDE3B; PDE4 isotypessuch as PDE4A, PDE4B and PDE4D and PDE7 isotypes such as PDE7A, amongother PDEs.

The present invention further provides an antisense oligonucleotideeffective in the compositions and methods of the present inventionhaving a sequence selected from the group consisting of; SEQ. ID NO.: 1;SEQ. ID NO.: 2; SEQ. ID NO.: 3; SEQ. ID NO.: 4; SEQ. ID NO.: 5; SEQ. IDNO.: 6; SEQ. ID NO.: 7; SEQ. ID NO.: 8; SEQ. ID NO.: 9; SEQ. ID NO.: 10;SEQ. ID NO.: 11; SEQ. ID NO.: 12; SEQ. ID NO.: 13; SEQ. ID NO.: 14; SEQ.ID NO.: 15; SEQ. ID NO.: 16; SEQ. ID NO.: 17; SEQ. ID NO.: 18; SEQ. IDNO.: 19; SEQ. ID NO.: 20; SEQ. ID NO.: 21; SEQ. ID NO.: 22; SEQ. ID NO.:23; SEQ. ID NO.: 27; SEQ. ID NO.: 28; SEQ. ID NO.: 33; SEQ. ID NO.: 34;SEQ. ID NO.: 35; SEQ. ID NO.: 36; SEQ. ID NO.: 24; SEQ. ID NO.: 25; SEQ.ID NO.: 26; SEQ. ID NO.: 29; SEQ. ID NO.: 30; SEQ. ID NO.: 31; SEQ. IDNO.: 32 (Table 1a-f).

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive description of severalpreferred embodiments made with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to thefollowing description and to the Figures in which:

FIG. 1 illustrates 6 gels of semi-quantitative PCR showing theexpression profile of different phosphodiesterases (PDEs) in structural(A549, bronchial smooth muscle cells (BSMC), NCI—H292); immune cells(peripheral blood mononuclear cells, PBMC) and in tissue (Lung, Brain).3A=PDE3A; 3B=PDE3B; 7A PDE7A; 7B=PDE7B; 4A PDE4A; 4B=PDE4B; 4C=PDE4C;4D=PDE4D; P=PBGD (PORPHOBILINOGEN DEAMINASE); G=GAPDH (Glyceraldehyde3-phosphate dehydrogenase); (−)=Amplification of RNA without reversetranscription; MW=100 bp DNA ladder;

FIG. 2 illustrates 7 gels of semi-quantitative PCR showing theexpression profile of PDE7A isotypes in structural (A549, NHBE, BSMC)and non-structural (Eol-1, Hut-78, Jurkat, and U937) cells that arerelated to potentially important cells in the lung inflammatoryresponse. 7A1=PDE7A1; 7A2=PDE7A2; 7A3=PDE7A3; G=GAPDH, (−)=Amplificationof RNA without reverse transcription and MW=100 bp DNA ladder;

-   -   A.

FIG. 3 illustrates 8 gels of semi-quantitative PCRs showing efficacy ofsome of the 1700 antisense series at decreasing PDE7A mRNA expression inEol-1 and U937 cells. C=no antisense; Top-1701, Top-1702, Top-1703,Top-1706, Top-1707 and Top-1726=PDE7A-directed antisenses; 100 bp=DNAladder; (0), (10) and (20)=antisense concentration in uM (micro-molar);

FIG. 4 illustrates 6 gels of semi-quantitative PCRs showing efficacy ofsome of the 1700 antisense series at decreasing PDE7A mRNA expression inJurkat and Hut-78 cells. C=no antisense; Top-1702, Top-1703, Top-1706,Top-1707 and Top-1726=PDE7A-directed antisenses; 100 bp=DNA ladder; (0),(10) and (20)=antisense concentration in uM (micro-molar);

FIG. 5 illustrates 6 gels of semi-quantitative PCRs showing efficacy ofsome of the 1700 antisense series at decreasing PDE7A mRNA expression inBSMC and A549 cells. C1 and C2=no antisense; Top-1702, Top-1703, andTop-1706=PDE7A-directed antisenses; MW=100 bp DNA ladder; (0), and(20)=antisense concentration in uM (micro-molar);

FIG. 6 illustrates 6 gels of semi-quantitative PCRs showing efficacy ofsome of the 1300 antisense series at decreasing PDE3A mRNA expression inA549 and U937 cells. C=no antisense; Top-1301, Top-1302, Top-1303,Top-1307, Top-1308, Top-1309 and Top-1311=PDE3A-directed antisenses; 100bp=DNA ladder; (0), (10) and (20)=antisense concentration in uM(micro-molar);

FIG. 7 illustrates a gel of semi-quantitative PCRs showing the efficacyof the 1300 antisense series at decreasing PDE3A mRNA expression in BSMCcells. C1 and C2=no antisense; Top-1301, Top-1302, Top-1303, Top-1307,Top-1308, Top-1309 and Top-1311=PDE3A-directed antisenses; 100 bp=DNAladder; (0), and (20)=antisense concentration in uM (micro-molar);

FIG. 8 illustrates semi-quantitative PCRs showing the relative efficacyof the antisense oligonucleotides of the 1300 and the 1700 antisenseseries at decreasing MMP-1, MMP-2, MMP-3, MMP-12, TIMP-1, COX-1, IL-6,IL-7, IL-8, IL-15, TNF-alpha in BSMC cells. C=no antisense; Top-1301,Top-1303, Top-1308, and Top-1311=PDE3A-directed antisenses; Top-1702,Top-1703, and Top-1706=PDE7A-directed antisenses; MW=100 bp DNA ladder;

FIG. 9 illustrates a gel of semi-quantitative PCRs showing the efficacyand the concept of multiple gene knock down of PDE3A and PDE7A mRNAexpression by combinations of oligos from the 1700 and 1300 series inU937 cells. C=no antisense; 1=Top-1301; 2=Top-1303; 3=Top-1307;4=Top-1311; 5=Top-1702; 6=Top-1703; 7=Top-1706; 8=Top-1702 and 1301;9=Top-1703 and Top-1301; 10=Top-1706 and Top-1301; 11=Top-1702 andTop-1303; 12=Top-1703 and Top-1303; 13=1706 and 1303; 14=Top-1702 andTop-1307; 15=Top-1703 and Top-1307; 16=Top-1706 and Top-1307;17=Top-1703 and Top-1311; 18=Top-1706 and Top-1311; MW=100 bp DNAladder. The combinations 9 and 13 are the ones that are effective atmultiple gene knock down;

FIG. 10 illustrates semi-quantitative PCRs showing the relative efficacyand concept of multiple gene knock down of the 1300 and the 1700antisense series at modulating the expression level of PDE4B, IL-10,IL-15, MMP-1, MMP-2, MMP-8, TIMP-1 in U937 cells. C=no antisense;Top-1301, Top-1303, Top-1307 and Top-1311=PDE3A-directed antisenses;Top-1703, and Top-1706=PDE7A-directed antisenses; MW=100 bp DNA ladder.1=Top-1301; 2=Top-1303; 3=Top-1703; 4=Top-1706; 5=Top-1301 and Top-1703;6=Top-1303 and 1706; 7=Top-1311 and Top-1703; 8=Top-1307 and Top-1703;

FIG. 11 shows the results of a functional assay for human PDE antisenses(AS) and their relative efficacy of multiple gene knock down at thelevel of TNF-alpha protein. The test assessed the effects of antisenseoligonucleotides directed against different isotypes of PDEs onTNF-alpha production in human PBMC stimulated with PHA. PBMC weretransfected with one AS, PDE3B AS: Top-1360 (SEQ. ID NO.: 19)=3B; PDE4BAS Top-1437 (SEQ. ID NO.: 28)=4B; PDE4D AS Top-1498 (SEQ. ID NO.:36)=4D; PDE4A AS Top-1413 (SEQ. ID NO.: 23)=4A. PBMC were alsotransfected with combinations of two AS, 3B/4B=Top-1360 (SEQ. ID NO.:19)+Top-1437 (SEQ. ID NO.: 28); 3B/4D=Top-1360 (SEQ. ID NO.:19)+Top-1498 (SEQ. ID NO.: 36); 4A/4D=Top-1413 (SEQ. ID NO.:23)+Top-1498 (SEQ. ID NO.: 36); 4B/4D=Top-1437 (SEQ. ID NO.:28)+Top-1498 (SEQ. ID NO.: 36). Mismatch ASs (3Bmi, 4Ami, 4Bmi and 4Dmi)were also included in this study. PHA challenged but non transfectedPBMC were considered as controls;

FIG. 12 shows the multiple target inhibition and synergistic effect ofcombined antisense treatment targeting both PDE4B and PDE7A. Mice (8 pergroup) were nasally instilled with 50 mg Top-2437 (targeting PDE4B), 50mg Top-2713 (targeting PDE7A) or a combination of both antisenses (50 mgof each). Control mice received the same amount of control antisenseoligonucleotide. Real-time PCR analysis was performed for (A) PDE4B, (B)PDE7A and (C) PDE4A expression, normalized to HPRT (reference gene) 16 hafter instillation. Data represent % of inhibition for each mouse(relative to control antisense treated mice) of PDE4B expression, PDE7Aexpression and PDE4A expression. Bars indicate mean % of inhibition;

FIG. 13 shows the effect of specific inhibition by antisenseoligonucleotides targeted at the PDE4B mRNA on LPS-induced lunginflammation and on PDE mRNA expression. A) Effect of the inhibition ofPDE4B on the expression of other mRNA for PDEs. The expression levels ofdifferent PDE mRNAs in TOP2430 treated mice were assessed by real-timePCR 6 h after LPS exposure. % of inhibition were determined by comparingthe expression levels obtained to the expression levels measured in micethat received the control antisense oligonucleotide. Values shownrepresent means +/− SD (n=8−9). B) Inhibition of the cellular influx inthe lung of LPS-exposed mice pre-treated with TOP2430. Differential cellcount of BAL cells was performed 6 h after LPS exposure. Values shownrepresent means +/− SD (n=8−9). C) Effect on TNF-alpha release in BAL.TNF-alpha release in TOP2430 treated mice was measured by ELISA and % ofinhibition were determined by comparing TNF-alpha values obtained afterTOP 2430 to the values obtained in mice treated with a control antisenseoligonucleotide; and

FIG. 14 shows the effect of specific inhibition by an antisenseoligonucleotide targeting the mouse PDE4B mRNA on cigarettesmoke-induced lung inflammation. A) Bronchoalveolar lavage cell counts24 h and 48 h after cigarette smoke exposure. Values are mean +/− SD.PMN=neutrophils; AM=macrophages. B) TNF-alpha mRNA expression in lungtissue of mice exposed to cigarette smoke. The expression levels ofTNF-alpha mRNA were assessed by real-time PCR on cDNA prepared from RNAisolated from whole lungs 48 h after cigarette smoke exposure (3 mice).Control mice were sham smoked (5 mice). Values are mean +/− SD ofTNF-alpha expression relative to HPRT. C) TNF-alpha and PDE4B mRNAexpression in mice pre-treated with TOP2430 (4 mice) or controlantisense (5 mice) and exposed to cigarette smoke 3 h later. Theexpression levels of TNF-alpha and PDE4B mRNA were assessed by real-timePCR on cDNA prepared from RNA isolated from whole lungs 48 h aftercigarette smoke exposure. Values are mean +/− SD of TNF-alpha or PDE4Bexpression relative to HPRT.

BRIEF DESCRIPTION OF THE TABLES

Table 1a identifies human PDE7A oligonucleotide antisenses in accordancewith the present invention;

Table 1b identifies the human PDE3A oligonucleotide antisenses inaccordance with the present invention;

Table 1c identifies the human PDE3B oligonucleotide antisenses inaccordance with the present invention;

Table 1d identifies the human PDE4A oligonucleotide antisenses inaccordance with the present invention;

Table 1e shows the human PDE4B oligonucleotide antisenses in accordancewith the present invention;

Table 1f shows the human PDE4D oligonucleotide antisenses in accordancewith the present invention;

Table 2a and 2b show human oligonucleotide primers used in standard PCR;

Table 2c shows human oligonucleotide primers used in real time PCR;

Table 2d shows mouse oligonucleotide primers used in real time PCR;

Table 3a and 3b show human PDE expression pattern in cell lines, primarycells and tissues;

Table 4a shows a summary of human PDE oligonucleotide antisenses primaryscreening in A549 cell line and in human PBMC;

Table 4b shows human PDE single antisenses with multiple gene knock downeffects; and

Table 5 shows mouse PDE oligonucleotide antisenses primary screening invivo;

DETAILED DESCRIPTION OF THE INVENTION

The invention herein relates to antisense oligonucleotide-basedcompounds, therapeutic compositions and methods for the treatment ofdisease associated with reduced cellular cAMP and/or disease associatedwith elevated levels of at least one PDE. The invention is aimed atincreasing the level of cAMP in cells, while decreasing the level ofpro-inflammatory mediators as well as enzymes that are released byinflammatory cells.

To obtain these effects, the present invention utilizes, in one of itsaspects, what is herein referred to as “multiple gene knock down”.Multiple gene knock down refers to the inhibition or downregulation ofmultiple genes by either a single antisense oligonucleotide compound inaccordance with the present invention which downregulates not only theisoenzyme gene that it is directed against but also a related gene(s),or by a combination of at least two antisense oligonucleotide compoundswhich each downregulate a different isoenzyme gene.

Diseases associated with reduced levels of cellular cAMP includediseases in which there is increased levels of at least onecAMP-specific PDE (i.e. a PDE-related disease).

The term “downregulate” is used herein to refer to at least partialinhibition of the expression of a gene. In accordance with theinvention, an antisense oligonucleotide compound downregulates orinhibits a gene that it is directed against, i.e. a gene to which theantisense oligonucleotide compound exhibits sequence complementaritysufficient to cause inhibition.

The term “related genes” refers to other genes that may also play a rolein the pathophysiology of disease associated with reduced cellular cAMPand/or increased levels of at least one PDE but to which theoligonucleotide antisense compound is not complementary, either wholelyor partially. Such genes include, but are not limited to, genes thatencode PDE enzymes or isotypes, mediators, for example, cytokines andchemokines and genes that encode enzymes. Examples of mediators includeIL-6, IL-7, IL-8, IL-15 and TNF-alpha. Examples of appropriate enzymesinclude, but are not limited to, matrix metalloproteinases (MMPs), suchas MMP-1, MMP-2, MMP-3, MMP-9 and MMP-12.

In accordance with the present invention, antisense oligonucleotidecompounds are herein defined as oligonucleotides, naturally occurring ormodified, preferably nuclease resistant, that exhibit a complementarityto DNA or mRNA coding for a particular target protein such that they arecapable of interfering with the transcription or translation of the mRNAand/or induce RNase or RNase-like activity and thereby function toreduce expression of the target protein. Expression of the targetprotein is reduced when the oligonucleotide compound hybridizes to thetarget DNA or mRNA, thereby interfering/preventing itstranscription/translation. The present oligonucleotide compounds, thus,must be sufficiently complementary to the nucleic acid to which it isdirected in order to hybridize thereto. Thus, although in a mostpreferred embodiment, the oligonucleotide compounds are 100%complementary to the nucleic to which they are directed, 100%complementarity is not necessary for hybridization to occur between anoligonucleotide compound and the nucleic acid to which it is directed,as one of skill will appreciate.

The invention herein also relates to modifications to an antisenseoligonucleotide(s) that do not significantly adversely effect theiractivity to reduce or inhibit expression of a target protein, but whichmay enhance this activity. The terms “nucleic acid” and “nucleic acidmolecule” as used interchangeably herein, refer to a molecule comprisedof nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both.The term includes monomers and polymers of ribonucleotides anddeoxyribonucleotides, with the ribonucleotide and/ordeoxyribonucleotides being connected together, in the case of thepolymers, via 5′ to 3′ linkages. However, linkages may include any ofthe linkages known in the nucleic acid synthesis art including, forexample, nucleic acids comprising 5′ to 2′ linkages. The nucleotidesused in the nucleic acid molecule may be naturally occurring or may besynthetically produced analogues that are capable of forming base-pairrelationships with naturally occurring base pairs. Examples ofnon-naturally occurring bases that are capable of forming base-pairingrelationships include, but are not limited to, aza and deaza pyrimidineanalogues, aza and deaza purine analogues, and other heterocyclic baseanalogues, wherein one or more of the carbon and nitrogen atoms of thepurine and pyrimidine rings have been substituted by heteroatoms, e.g.,oxygen, sulfur, selenium, phosphorus, and the like.

The present antisense oligonucleotide compounds may also be modified byinsertion or deletion of 1 or more bases without significant adverseeffect to their activity. In particular, the addition or deletion ofbases at the terminal ends of the oligonucleotides that exhibit 100%complementation to the gene they are directed against can generally bemade without significant loss of inhibitory activity. Such modificationsmay be made in order to increase activity or to provide enhancedstability of the oligonucleotide. In addition, substitution of 1 or morebases in the present antisense oligonucleotide compounds may also bemade without adverse effect to activity, for example, substitution ofpurine with another purine (adenine, guanine) and pyrimidine withpyrimidine (cytosine, thymine, uracil).

Antisense oligonucleotide compounds in accordance with the presentinvention also include siRNAs (small interfering RNAs) and the RISCs(RNA-induced silencing complexes) containing them that result from theRNAi (RNA interference) approach. The RNA interference (RNAi) approach,which has been described recently, is considered as a new tool for theinhibition of target gene expression. As already known some years ago,RNAi is based on an ancient anti-viral defence mechanism in lowereukaryotes. It is induced by double-stranded RNA and its processing to21-23 nt small interfering RNAs (siRNAs), which cause the degradation ofhomologous endogenous mRNA after hybridizing to the target mRNA in asingle stranded fashion with the assistance of the RISC complex. The wayRNAi works is still to be fully elucidated, but it already serves as afirst-choice approach to generate loss-of-function phenotypes among abroad variety of eukaryotic species, such as nematodes, flies, plants,fungi and mammals.

Antisense oligonucleotide compounds in accordance with the presentinvention also include ribozymes and short nucleotide sequences, singleor double stranded, RNA or DNA, which may incorporate chemicalmodifications as described above, capable of inhibiting genetranscription and/or translation in vitro and/or in vivo.

The compositions and methods of the present invention, in one aspect,while not to be bound by a particular mode of action, appear to act by amechanism that has been termed “multiple gene knock down”. Multiple geneknock down in the context of the present invention refers to:

1) an antisense oligonucleotide compound that downregulates not only thegene that it is directed against but also downregulates other relatedgenes; and2) a combination of at least 2 antisense oligonucleotide compounds eachcapable of downregulating a gene, each being present at a concentrationat which the antisense oligonucleotide compound is practicallyineffective on its own to downregulate a gene it is directed against,the combination leading to significant downregulation of both genes thatthe antisense oligonucleotides are directed against and may alsodownregulate other related genes.

In one aspect, the present invention provides a pharmaceuticalcomposition for treating and/or preventing disease associated withreduced cellular cAMP and/or elevated levels of at least one PDE, saidcomposition comprising a pharmaceutically acceptable carrier and atleast two antisense oligonucleotide compounds, each oligonucleotidecompound being directed against at least a portion of a different targetPDE-encoding gene, each oligonucleotide compound being capable ofdownregulating the target gene it is directed against, eacholigonucleotide compound being present at a concentration at which itexhibits less than 20% inhibition of its target PDE gene, thecombination exhibiting greater than 20% inhibition and at least doublingthe inhibition of at least one of the target genes and optionally otherrelated genes.

As one of skill in the art will appreciate, the combination ofoligonucleotide compounds in accordance with the present invention atvery low concentrations, for example, concentrations with exhibit lessthan 1% inhibition of a target PDE gene, may not combine to exhibit aninhibition of at least 20%. The lowest concentration at which eacholigonucleotide compound can be used to form a combination in accordancewith the present invention will vary, and this concentration may be thatat which 0.5% inhibition is achieved, or that concentration at which 1%to 5% inhibition is achieved.

The present invention provides a pharmaceutical composition for treatingand/or preventing a disease associated with reduced cellular cAMP and/orelevated levels of at least one PDE, said composition comprising apharmaceutically acceptable carrier and at least two antisenseoligonucleotides that are each capable of downregulating a differenttarget gene, at least one of the oligonucleotides being present at aconcentration at which it is practically ineffective on its own, e.g. aconcentration exhibiting less than 20% inhibition of the target gene,the combination of the oligonucleotides exhibiting more than 20%inhibition and at least doubling the inhibition of at least one of thetarget genes. and optionally other related genes.

The present invention further provides the use of a combination of atleast two antisense oligonucleotide compounds for treating and/orpreventing disease associated with reduced cellular cAMP, such asrespiratory inflammatory disease, each oligonucleotide compound beingdirected against a gene coding for a different PDE isotype, eacholigonucleotide compound being capable of downregulating the gene it isdirected against, the oligonucleotide compounds being present at aconcentration at which each oligonucleotide is practically ineffectiveon its own to downregulate the gene it is directed against, thecombination of the at least two oligonucleotide compounds beingeffective to downregulate at least one of the genes the oligonucleotidecompounds are directed against and optionally other related genes.

The present invention further provides a method of treating and/orpreventing disease associated with reduced cellular cAMP, such asinflammatory respiratory disease, the method comprising administering toa subject at least two antisense oligonucleotide compounds each beingcapable of downregulating a different target PDE gene, theoligonucleotide compounds being administered at a concentration at whicheach oligonucleotide compound is practically ineffective on its own todownregulate its target gene, for example, a concentration exhibitingless than 20% inhibition of the target gene, the combination of theoligonucleotide compounds being effective to downregulate at least oneof the genes the oligonucleotide compounds are directed against andoptionally other related genes.

The present invention further provides the use of a pharmaceuticalcomposition as described above for the manufacture of a medicament forthe treatment and/or prevention of a disease associated with reducedcellular cAMP or increased cAMP-specific PDE levels (PDE-relateddisease) such as inflammatory disease. Inflammatory disease to which thepresent methods and compositions are directed is generally defined asdiseases in which inflammatory cells and/or mediators are present.Examples of inflammatory diseases include, but are not limited to,multiple sclerosis, contact dermatitis, allergic and non-allergic eyediseases, rheumatoid arthritis, septic shock, osteoporosis and cognitivedisorders. Inflammatory respiratory disease to which the present methodsand compositions are directed is generally defined as diseases of therespiratory tract and lungs in which inflammatory cells and mediatorsare present. Examples of inflammatory respiratory disease include, butare not limited to, COPD, asthma, eosinophilic cough, bronchitis, acuteand chronic rejection of lung allograft, sarcoidosis, pulmonaryfibrosis, rhinitis and sinusitis.

The present invention further provides the use of a pharmaceuticalcomposition as described above for the manufacture of a medicament forthe treatment and/or prevention of a disease where decreased cyclic AMPis involved in the physiopathology.

The present invention further provides methods for modifying antisenseoligonucleotides so that it may reach the target nucleotide and/or bemore effective against the target gene for the treatment and/orprevention of inflammatory respiratory diseases.

The present invention further provides a formulation, including thecomposition described above, the formulation being systemic and/ortopical.

The present invention further provides an in vivo method of delivering apharmaceutical composition to a target polynucleotide, comprisingadministering to a subject the composition as described above incombination with a surfactant that permits the composition to reach thetarget gene(s).

Preferably, the subjects or patients that may be treated using theantisense oligonucleotide compounds of the present invention include,but are not limited to, invertebrates, vertebrates, birds, mammals suchas pigs, goats, sheep, cows, dogs, cats, and particularly humans.Oligonucleotide compounds in accordance with the present invention aredesigned to be appropriate to the particular animal to be treated. Inparticular, the sequence of the antisense oligonucleotide compounds willvary with the subject being treated given inter-species genomedifferences.

The present invention further provides antisense oligonucleotideseffective against, e.g. capable of inhibiting the expression of, the PDEfamily of enzymes, including, but not limited to, PDE3, PDE4 and PDE7.As will be appreciated by one of skill in the art, each PDE subtype mayalso includes isotypes. For example, PDE3 subtype include the PDE3A andPDE3B isotypes; the PDE4 subtype includes isotypes PDE4A, PDE4B, PDE4Cand PDE4D; and the PDE7 subtype includes isotypes PDE7A1, PDE7A2, PDE7A3and PDE7B.

When used herein the term “concentration at which the antisenseoligonucleotide compound is practically ineffective” refers to the useof each oligonucleotide compound, when taken alone, at a concentrationat which practically no downregulation is observed for the gene againstwhich the oligonucleotide compound is directed (at a concentration atwhich less than 20% inhibition of the target gene is exhibited). This isin contrast to the effect of the combination of at least twooligonucleotide compounds in accordance with the present invention, eachpresent at a concentration at which they are ineffective alone todownregulate the gene they are directed against (at a concentration atwhich less than 20% inhibition is exhibited), this combinationexhibiting greater than 20% inhibition and being effective to at leastdouble the inhibition of at least one of the genes against which theoligonucleotide compounds are directed.

The comparison of effectiveness of a single oligonucleotide isillustrated in, but not limited to, FIGS. 13 and 14 and table 4b and 5.For example in FIG. 13 A TOP 2430 which is directed against mouse PDE4Binhibits in vivo PDE4A, 4B, 4D and 7A but has no effect on PDE3B.

The comparison of effectiveness of combinations of at least 2oligonucleotide compounds is illustrated in, but not limited to, FIGS.9, 10, 11 and 12. For example in FIG. 9 the combination of Top-1703 (SeqID No. 2) and Top-1301 (SEQ. ID NO.: 10) or Top-1706 (SEQ. ID NO.: 3)and Top-1303 (SEQ. ID NO.: 12) show a greater effect at decreasingexpression of both PDE3A and PDE7A than either of the oligonucleotideshad on either PDE3A or PDE7A expression when used alone. In FIG. 11 thecombination of Top-1360 (SEQ. ID NO.: 19) and Top-1437 (SEQ. ID NO.: 28)or Top-1360 (SEQ. ID NO.: 19) and Top-1498 (SEQ. ID NO.: 36) or Top-1413(SEQ. ID NO.: 23) and Top-1498 (SEQ. ID NO.: 36) or Top-1437 (SEQ. IDNO.: 28) and Top-1498 (SEQ. ID NO.: 36), show a greater effect atdecreasing TNF-alpha expression and release in human PBMC than either ofthe oligonucleotides had on TNF-alpha when used alone. In FIG. 12 invivo nasal instillation of mice with the combination of Top-2437 (PDE4B)and Top-2713 (PDE7A) show a greater effect at decreasing both mousePDE4B and PDE7A expression than either of the oligonucleotides had oneither PDE4B or PDE7A expression when used alone at low concentrations.

Many combinations of antisense oligonucleotide compounds may be used inaccordance with the present invention, the combinations leading tomultiple gene knock down as described above. Examples of combinations ofantisense oligonucleotides may include, but are not limited to, at leastone oligonucleotide compound directed against PDE3 with at least oneoligonucleotide compound directed against PDE7. An alternative exampleof a combination may include at least one oligonucleotide directedagainst PDE4 and at least one oligonucleotide directed against PDE7. Analternative example of a combination may include at least oneoligonucleotide directed against PDE3, at least one oligonucleotidedirected against PDE4 and at least one oligonucleotide directed againstPDE7. Such antisense oligonucleotide compounds may be chosen from, butare not limited to, the oligonucleotides provided in Tables 1a-f.

The term “oligonucleotide” as used herein refers to a nucleic acidmolecule comprising from about 1 to about 100 nucleotides, morepreferably from 1 to 80 nucleotides, and even more preferably from about4 to about 35 nucleotides. The term “oligonucleotide” also includesmodified oligonucleotides and oligonucleotide compounds as set outabove.

The terms “modified oligonucleotide” and “modified nucleic acidmolecule” as used herein refer to nucleic acids, includingoligonucleotides, with one or more chemical modifications at themolecular level of the natural molecular structures of all or any of thenucleic acid bases, sugar moieties, internucleoside phosphate linkages,as well as molecules having added substituents, such as diamines,cholesteryl or other lipophilic groups, or a combination ofmodifications at these sites. The internucleoside phosphate linkages canbe phosphodiester, phosphotriester, phosphoramidate, siloxane,carbonate, carboxymethylester, acetamidate, carbamate, thioether,bridged phosphoranidate, bridged methylene phosphonate,phosphorothioate, methylphosphonate, phosphorodithioate, bridgedphosphorothioate and/or sulfone internucleotide linkages, or 3′-3′,2′-5′ or 5′-5′ linkages, and combinations of such similar linkages (toproduce mixed backbone modified oligonucleotides). The modifications canbe internal (single or repeated) or at the end(s) of the oligonucleotidemolecule and can include additions to the molecule of theinternucleoside phosphate linkages, such as cholesteryl, diaminecompounds with varying numbers of carbon residues between amino groupsand terminal ribose, deoxyribose and phosphate modifications whichcleave or cross-link to the opposite chains or to associated enzymes orother proteins. Electrophilic groups such as ribose-dialdehyde may becovalently linked with an epsilon amino group of the lysyl-residue ofsuch a protein. A nucleophilic group such as n-ethylmaleimide tetheredto an oligomer could covalently attach to the 5′ end of an mRNA or toanother electrophilic site. The term modified oligonucleotides alsoincludes oligonucleotides comprising modifications to the sugar moietiessuch as 2′-substituted ribonucleotides, or deoxyribonucleotide monomers,any of which are connected together via 5′ to 3′ linkages. Modifiedoligonucleotides may also be comprised of PNA or morpholino modifiedbackbones where target specificity of the sequence is maintained.

Optionally, the presently described oligonucleotides may be formulatedwith a variety of physiological carrier molecules. The presentlydescribed oligonucleotides may also be complexed with molecules thatenhance their ability to enter the target cells. Examples of suchmolecules include, but are not limited to, carbohydrates, polyamines,amino acids, peptides, lipids, and molecules vital to cell growth. Forexample, the oligonucleotides may be combined with a lipid, theresulting oligonucleotide/lipid emulsion, or liposomal suspension may,inter alia, effectively increase the in vivo half-life of theoligonucleotide.

Alternatively, oligonucleotides directed at PDE targets may also beprotonated/acidified to function in a dual role as phosphodiesteraseinhibitors and antibacterial agents. Accordingly, another embodiment ofthe presently described invention is the use of a PDE modulatingtherapeutic oligonucleotide that is additionally protonated/acidified toincrease cellular uptake, improve encapsulation in liposomes, so it canalso serve as an antibiotic. Additionally, the oligonucleotide may becomplexed with a variety of well established compounds or structuresthat, for instance, further enhance the in vivo stability of theoligonucleotide, or otherwise enhance its pharmacological properties(e.g., increase in vivo half-life, reduce toxicity, etc.).

The term “nucleic acid backbone” as used herein refers to the structureof the chemical moiety linking nucleotides in a molecule. This mayinclude structures formed from any and all means of chemically linkingnucleotides. A modified backbone as used herein includes modificationsto the chemical linkage between nucleotides, as well as othermodifications that may be used to enhance stability and affinity, suchas modifications to the sugar structure. For example an [alpha]-anomerof deoxyribose may be used, where the base is inverted with respect tothe natural [beta]-anomer. In a preferred embodiment, the 2′-OH of thesugar group may be altered to 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl), whichprovides resistance to degradation without comprising affinity.

The term “acidification” and “protonation/acidification” as usedinterchangeably herein refers to the process by which protons (orpositive hydrogen ions) are added to proton acceptor sites on a nucleicacid. The proton acceptor sites include the amine groups on the basestructures of the nucleic acid and the phosphate of the phosphodiesterlinkages. As the pH is decreased, the number of these acceptor siteswhich are protonated increases, resulting in a more highlyprotonated/acidified nucleic acid.

The term “protonated/acidified nucleic acid” refers to a nucleic acidthat, when dissolved in water at a concentration of approximately 16A260 per ml, has a pH lower than physiological pH, i.e., lower thanapproximately pH 7. Modified nucleic acids, nuclease-resistant nucleicacids, and antisense nucleic acids are meant to be encompassed by thisdefinition. Generally, nucleic acids are protonated/acidified by addingprotons to the reactive sites on a nucleic acid, although othermodifications that will decrease the pH of the nucleic acid can also beused and are intended to be encompassed by this term.

The term “end-blocked” as used herein refers to a nucleic acid with achemical modification at the molecular level that prevents thedegradation of selected nucleotides, e.g., by nuclease action. Thischemical modification is positioned such that it protects the integralportion of the nucleic acid, for example the coding region of anantisense oligonucleotide. An end block may be a 3′ end block or a 5′end block. For example, a 3′ end block may be at the 3′-most position ofthe molecule, or it may be internal to the 3′ ends, provided it is 3′ ofthe integral sequences of the nucleic acid.

The term “substantially nuclease resistant” refers to nucleic acids thatare resistant to nuclease degradation, as compared to naturallyoccurring or unmodified nucleic acids. Modified nucleic acids of theinvention are at least 1.25 times more resistant to nuclease degradationthan their unmodified counterpart, more preferably at least 2 times moreresistant, even more preferably at least 5 times more resistant, andmost preferably at least 10 times more resistant than their unmodifiedcounterpart. Such substantially nuclease resistant nucleic acidsinclude, but are not limited to, nucleic acids with modified backbonessuch as phosphorothioates, methylphosphonates, ethylphosphotriesters,2′-0-methylphosphorothioates, 2′-O-methyl-p-ethoxy ribonucleotides,2′-O-alkyls, 2′-O-alkyl-n(O-alkyl), 3′-O-alkyls, 3′-O-alkyl-n(O-alkyl),2′-fluoros, 2′-deoxy-erythropentofuranosyls, 2′-O-methylribonucleosides, methyl carbamates, methyl carbonates, inverted bases(e.g., inverted T's), or chimeric versions of these backbones.

The term “substantially acid resistant” as used herein refers to nucleicacids that are resistant to acid degradation as compared to unmodifiednucleic acids. Typically, the relative acid resistance of a nucleic acidwill be measured by comparing the percent degradation of a resistantnucleic acid with the percent degradation of its unmodified counterpart(i.e., a corresponding nucleic acid with “normal” backbone, bases, andphosphodiester linkages). A nucleic acid that is acid resistant ispreferably at least 1.5 times more resistant to acid degradation, atleast 2 times more resistant, even more preferably at least 5 times moreresistant, and most preferably at least 10 times more resistant thantheir unmodified counterpart.

The terms “PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, PDE7Boligonucleotide” as used herein each refer to an oligonucleotide that istargeted, respectively, to sequences that affect PDE3A, PDE3B, PDE4A,PDE4B, PDE4C, PDE4D, PDE7A, PDE7B expression or activity. These include,but are not limited to, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE7A,PDE7B DNA coding sequences, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D,PDE7A, PDE7B DNA promoter sequences, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C,PDE4D, PDE7A, PDE7B DNA enhancer sequences, mRNA encoding PDE3A, PDE3B,PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, PDE7B, and the like.

As discussed above, one embodiment of the present invention providesantisense oligonucleotides targeted to sequences that affect PDE3A,PDE3B, PDE4A, PDE4B, PDE4D, PDE7A, expression or activity. In oneembodiment the antisense oligonucleotides may have one of the sequencesidentified in Tables 1a-f. In another embodiment, the antisenseoligonucleotide may comprise fragments or variants of these sequences,as will be understood by a person skilled in the art, that may alter theoligonucleotide make-up and/or length, but which maintains or increasesthe activity of the oligonucleotide to downregulate gene expression. Inanother embodiment the present invention provides for combinations of atleast two antisense oligonucleotides having the sequences identified inTables 1a-f.

The terms “treatment”, “treating”, “therapy” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or amelioration of an adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in asubject as previously defined, particularly a human, and includes:

(a) preventing a disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting a disease, i.e., arresting its development; or(c) relieving a disease, i.e., causing regression of the disease.

The term “pharmaceutically acceptable” as it is used herein with respectto carriers, surfactants and compositions refers to substances which areacceptable for use in the treatment of a subject patient that are nottoxic or otherwise unacceptable for administration by any of the routesherein described.

The invention is generally directed toward the treatment of subjects bythe administration of therapeutically effective amounts of antisenseoligonucleotide compounds in accordance with the present invention,including siRNA, ribozymes, short nucleotide sequences as single ordouble stranded including RNA and/or DNA that may be complementary to atarget nucleic acid, or may optionally be modified as described above,an RNA oligonucleotide having at least a portion of said RNAoligonucleotide capable of hybridizing with RNA to form anoligonucleotide-RNA duplex, or a chimeric oligonucleotide, that willdownregulate or inhibit the expression of an endogenous gene in vivo.

By “therapeutically effective” amount is meant a nontoxic but sufficientamount of an antisense oligonucleotide compound to provide the desiredtherapeutic effect. In the present case, that dose of antisenseoligonucleotide compound effective to relieve, ameliorate, or preventsymptoms of the condition or disease being treated, e.g. diseaseassociated with reduced cellular cAMP, PDE-related disease, inflammatorydisease such as inflammatory respiratory disease.

The pharmaceutical compositions provided herein comprise antisenseoligonucleotide compounds described above and one or morepharmaceutically acceptable surfactants. Suitable surfactants orsurfactant components for enhancing the uptake of the antisenseoligonucleotides of the invention have been previously described in U.S.Application Publication No. 2003/0087845, the contents of which areincorporated with respect to surfactants The application states thatsuitable surfactants “ . . . include synthetic and natural as well asfull and truncated forms of surfactant protein A, surfactant protein B,surfactant protein C, surfactant protein D and surfactant protein E,di-saturated phosphatidylcholine (other than dipalmitoyl),dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine; phosphatidic acid, ubiquinones,lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, cholinephosphate; as well as natural and artificial lamelar bodies which arethe natural carrier vehicles for the components of surfactant, omega-3fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid,non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants may be usedeither as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the antisense oligonucleotides (oligos).”

The antisense component of the present compositions, which includes thecombination of at least two antisense oligonucleotide compounds, may becontained in a pharmaceutical formulation within a lipid particle orvesicle, such as a liposome or microcrystal. As described in U.S. Pat.No. 6,025,339, the lipid particles may be of any suitable structure,such as unilamellar or plurilamellar, so long as the antisenseoligonucleotide is contained therein. Positively charged lipids such asN-[1-(2,3-dioleoyloxi) propyl]-N,N,N-trimethyl-ammoniumethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. See, e.g., U.S. Pat.Nos. 4,880,635 to Janoff et al.; 4,906,477 to Kurono et al.; 4,911,928to Wallach; 4,917,951 to Wallach; 4,920,016 to Allen et al.; 4,921,757to Wheatley et al.; etc.

The composition of the invention may be administered by any means thattransports the antisense oligonucleotide compound to the desired site,such as the lung. The antisense compounds disclosed herein may beadministered to the lungs of a patient by any suitable means, but arepreferably administered by inhalation of an aerosol comprised ofrespirable particles that comprise the antisense compound.

The composition of the present invention may be administered into therespiratory system as a formulation including particles of respirablesize, e.g. particles of a size sufficiently small to pass through thenose, mouth and larynx upon inhalation and through the bronchi andalveoli of the lungs. In general, respirable particles range from about0.5 to 10 microns in size. Particles of non-respirable size that areincluded in the aerosol tend to deposit in the throat and be swallowed,and the quantity of non-respirable particles in the aerosol ispreferably thus minimized. For nasal administration, a particle size inthe range of 10-500 micro-M (micro-meter)) is preferred to ensureretention in the nasal cavity.

Liquid pharmaceutical compositions of active compound (the antisenseoligonucleotide compound(s)) for producing an aerosol may be prepared bycombining the antisense compound with a suitable vehicle, such assterile pyrogen free water or phosphate buffered saline.

A solid particulate composition comprising the antisense compound mayoptionally contain a dispersant that serves to facilitate the formationof an aerosol as well as other therapeutic compounds. A suitabledispersant is lactose, which may be blended with the antisense compoundin any suitable ratio, e.g., a 1 to 1 ratio by weight.

The antisense compositions may be administered in ananti-bronchoconstriction, anti-allergy(ies) and/or anti-inflammatoryeffective amount, which amount depends upon the degree of disease beingtreated, the condition of the subject patient, the particularformulation, the route of administration, the timing of administrationto a subject, etc. In general, intracellular concentrations of theoligonucleotide of from 0.05 to 50.micro.M, or more particularly 0.2 to5.micro.M, are desirable. For administration to a mammalian patient suchas a human, a dosage of about 0.001, 0.01, 0.1, or 1 mg/Kg up to about50, or 100 mg/Kg or more is typically employed. However, other doses arealso contemplated. Depending on the solubility of the active compound inany particular formulation, the daily dose may be divided among one orseveral unit dose administrations.

The aerosols of liquid particles comprising the antisense compound maybe produced by any suitable means, such as with a nebulizer. Nebulizersare commercially available devices that transform solutions orsuspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of a compressed gas, typically air oroxygen, through a narrow venturi orifice or by means of ultrasonicagitation. Suitable formulations for use in nebulizers comprise theactive antisense oligonucleotide ingredient in a liquid carrier in anamount of up to 40% w/w preferably less than 20% w/w of the formulation.The carrier is typically water or a dilute aqueous alcoholic solution,preferably made isotonic with body fluids by the addition of, forexample, sodium chloride. Optional additives include preservatives ifthe formulation is not prepared sterile, for example, methylhydroxybenzoate, anti-oxidants, anti-bacterials, flavorings, volatileoils, buffering agents and emulsifiers and other formulationsurfactants.

The aerosols of solid particles comprising the active oligonucleotidecompound(s) and a pharmaceutically acceptable surfactant may likewise beproduced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles that are respirable, as explained above, andgenerate a volume of aerosol containing a predetermined metered dose ofa medicament at a rate suitable for human administration. The activeoligonucleotide ingredient typically comprises from 0.1 to 100 w/w ofthe formulation. A second type of illustrative aerosol generatorcomprises a metered dose inhaler. Metered dose inhalers are pressurizedaerosol dispensers, typically containing a suspension or solutionformulation of the active ingredient in a liquified propellant. Duringuse these devices discharge the formulation through a valve adapted todeliver a metered volume, typically from 10 to 150 .mu.l, to produce afine particle spray containing the active ingredient. Suitablepropellants include certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or hydrofluoroalkanes and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol, emulsifiers and other formulation surfactants, such asoleic acid or sorbitan trioleate, anti-oxidants and suitable flavoringagents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 1 to 150liters per minute.

The formulations of the present invention may be oral, intrabuccal,intrapulmonary, rectal, intrauterine, intratumor, intracranial, nasal,intramuscular, subcutaneous, intravascular, intrathecal, inhalable,transdermal, intradermal, intracavitary, implantable, iontophoretic,ocular, vaginal, intraarticular, otical, intravenous, intramuscular,intraglandular, intraorgan, intralymphatic, implantable, slow release orenteric coating formulations. The carriers used in the formulations maybe, for example, solid and/or liquid carriers.

Reference may be made to “Remington's Pharmaceutical Sciences”, 17thEd., Mack Publishing Company, Easton, Pa., 1985, for other carriers thatwould be suitable for combination with the present oligonucleotidecompounds to render compositions/formulations suitable foradministration to treat respiratory disease.

In a further aspect of the present invention, an article of manufactureis provided which includes packaging material contained within which isa pharmaceutically acceptable antisense oligonucleotide composition thatis therapeutically effective to treat conditions associated with reducedcellular cAMP, or elevated levels of PDE, including inflammatorydisease. In one embodiment, the composition comprises an antisenseoligonucleotide compound that is effective to inhibit a PDE gene, saidoligonucleotide compound being at least 50% complementary to the gene.In another aspect, the composition comprises at least 2 antisenseoligonucleotide compounds, each antisense oligonucleotide compound beingcapable of downregulating a different PDE gene, each antisenseoligonucleotide compound being present at a concentration at which theantisense oligonucleotide compound is practically ineffective on its ownto down-regulate the gene it is directed against, the combination of theantisense oligonucleotide compounds being effective to downregulate atleast one of the genes that the antisense oligonucleotides are directedagainst.

In one embodiment, the packaging material of the article comprises alabel which indicates that the composition can be used to treatinflammatory disease and may additionally include an indication that thedisease is one of multiple sclerosis, contact dermatitis, allergic andnon-allergic eye diseases, rheumatoid arthritis, septic shock,osteoporosis and cognitive disorders.

In another embodiment, the packaging material of the article comprises alabel which indicates that the composition can be used to treatinflammatory respiratory disease, and may additionally include anindication that the disease is one of COPD, asthma, eosinophilic cough,bronchitis, acute and chronic rejection of lung allograft, sarcoidosis,pulmonary fibrosis, rhinitis or sinusitis.

For the purposes of the present invention, the packaging material may beany suitable material for packaging a nucleotide-containing compositionin accordance with the present invention, including a bottle or othercontainer (either plastic or glass), a carton, a tube, or otherprotective wrapping. As will be appreciated, the packaging may vary withthe nature of the oligonucleotide composition, for example, a liquidformulation may be packaged differently than an aerosol formulation.

The present invention is illustrated in further detail by the followingnon-limiting examples.

Materials and Reagents

RPMI 1640 (Wisent, cat# 10040 CV); FBS (Fetal Bovine Serum, Wisent, cat#80150); Penicillin, Streptomycin (GIBCO, cat# 15140-122); HEPES (Wisent,cat# 26060CI) and L-glutamine (Gibco, cat# 25030-081); DMEM/F12 (Wisent,cat# 10090CV); Sodium Pyruvate (Wisent, cat# 25000-Ci); PBS Sterile(GIBCO, cat# 25030-081); Trypsin 0.25% and EDTA 0.01% (Wisent, cat#25-052-Ci); Hanks Balanced Salt Solution (HBSS) cellgro, cat#20021-cv;PHA (Phytohemaglutinin, Sigma, cat#L-9132, lot#: 073K8925) hEGF (humanrecombinant epidermal growth factor, cat-4230; BPE: clonetics cat#4009;hydrocortisone: clonetics cat#4031; epinephrine: clonetics cat#4221;transferring: clonetics cat#4205; insulin: clonetics cat#4021; retinoicacid: clonetics cat#4085; triiodothyronine: clonetics cat#4211;gentamicin/amphotericinB: clonetics cat#4081; human epithelial growthfactor (hEGF): clonetics cat#4230; Insulin, cat# cc-4021; hFGF (humanfibroblast growth factor) cat# cc-4068; SmBm Bullet kits (Cat# CC 3182,Clonetics). BEBM Bullet kits (Cat# CC 4175, Clonetics); Trizol:Invitrogen, cat#15596-018Dnase I: Fermentas, cat#EN0521; SuperscriptFirst-Strand Synthesis System for RT-PCR kit (Invitrogen,cat#11904-018); dNTPs: Invitrogen, cat# 10297-018; oligo (dT)₁₂₋₁₈:Invitrogen, cat#11904-018; Qiagen RNAeasy Mini Kit (Qiagen, Cat#74106);β-Mercaptoethanol (Sigma, Cat#M-6250); • 99% Ethanol (Commercialalcohols Inc., Brampton, Ontario, Canada); • QiaVac 24 Manifold (Qiagen,Cat#19403); •

Disposable Vacconnectors (Qiagen, Cat#19407); • DNAse I kit (Fermentas,Cat.# EN0521); • RiboGreen Quantification Reagent (Invitrogen-Molecularprobes, Cat # R-11490); Taq PCR core kit (Qiagen, cat#201223), •Light-Cycler Instrument version 1.5 (Roche, Cat# 3531414); • LCCapillaries for 20 ml reactions (Roche, cat# 1909339); • LC FastStartDNA Master SYBR Green 1 PLUS (100 ml) (Roche, Cat# 03752186001); K3EDTAtubes (Greiner Bio-one, cat#: 455036B110306); Ficoll (AmershamBiosciences; cat#: 17-1440-03); Human TNF-alpha ELISA kit, (BioSourceCat#CHC-1754, lot# 041703); Mouse TNF-alpha ELISA kit (BioSource,cat#cmc3013); ELISA plate reader (filter 450 nm, reference filter 650nm, Biorad, model 680); Escort II transfection reagent, (Sigma,cat#L6037), Lipopolysaccharide (SIGMA-Aldrich; cat.#L-4391, lot #083K4048, E. Coli 0111:B4); Xylazine (Anased), (Novopharm;cat.#02239093; lot no. KA09802D); Ketamine (Vetalar), (Bioniche;cat.#01989529; lot.#K033A); Hema-3 stain set (Fisher scientific Co.Cat#122-911, lot#999901); Polytron PT 1200 (Brinkmann Instruments);Alamar Blue, (Biosource cat#DAL1100), Human Lung Total RNA, (BDBioscience, cat#64092-1, lot#4030253); Human smooth muscle total RNA,(BD Bioscience, cat#cr2628, lot#3110321); Human brain total RNA, (BDBioscience, cat#64098-1, lot#4010842).

Cells

Human Bronchial smooth muscle cells (BSMC) (cat# CC 2576); normal humanbronchial epithelial cells (NHBE) (cat# CC 2540) were purchased fromClonetics. EOL-1 (Human acute myeloid “Eosinophilic” leukemia cell line;ATCC). U937 (Human histiocytic cell line; ATCC), HL-60 (Human acutepromyelocytic leukemia cell line; ATCC); A549 (Human lung carcinoma celllines; ATCC); Jurkat (Human acute T cell leukemia; ATCC) and Hut-78(Human cutaneous T lymphocyte lymphoma cell lines; ATCC). NCI—H292(Human mucoepidermoid pulmonary carcinoma cell line, ATCC); PBMC, (Humanperipheral blood mononuclear cells).

Cell Culture

EOL-1, U937, HL-60, Jurkat and NCI—H292 were cultured in RPMI 1640 with2 mM L-glutamine; 1.5 g/L sodium bicarbonate; 4.5 g/L glucose; 10 mMHepes; 1 mM sodium pyruvate; 10% FBS, Penicillin 100 U/mL, Streptomycin100 microg/mL.

Hut78 were cultivated in Iscove's modified medium with 4 mM L-glutamine;1.5 g/L sodium bicarbonate; 10% FBS, Penicillin 100 U/mL, Streptomycin100 mcg/mL.

A549 were cultivated in Ham's F12K with 2 mM L-glutamin; 1.5 g/L sodiumbicarbonate; 10% FBS, Penicillin 100 U/mL, Streptomycin 100 microg/mL.

Bronchial smooth muscle cells were cultured in 25 cm² flasks with SmBmmedium which contains: 0.5 microg/mL hEGF; 25 mg/ml gentamicin; 25microg/mL Amphotericin B; 2.5 mg/mL Insulin; hFGF 10 nanog/mL and FBS5%. Cell culture was performed for 10 days in order to obtain enoughcells for each experiment.

Normal human bronchial epithelial cells (NHBE) were cultured in 25 cm²flasks with BEBM medium that contained basal medium (500 mL) and thefollowing growth supplements: BPE: 2 mL; Hydrocortisone: 0.5 mL; hEGF:0.5 mL; Epinephrine: 0.5 mL; Transferrin: 0.5 mL; Insulin: 0.5 mL;Retinoic Acid: 0.5 mL; Triiodothyronin: 0.5 mL; GA-1000: 0.5 mL. Cellculture was performed for 10 days in order to obtain enough cells foreach experiment.

Cell Viability

Cell viability was systematically assayed using Alamar Blue test assuggested by the manufacturer.

Human Peripheral Blood Mononuclear Cells (PBMC)

PBMC were isolated by Ficoll-Hypaque density gradient centrifugation ofEDTA K3 blood from normal donors. PBMC were plated at 2×10⁶cells/mL/well in 12 well plates in RPMI 1640 cell culture mediumsupplemented with 10% heat inactivated FBS, Penicillin 100 U/mL,Streptomycin 100 microg/mL.

Antisense Treatment U937, EOL-1, Jurkat, Hut-78 Cell Lines:

Cells were harvested by centrifugation (5 minutes, 1500 RPM, at roomtemperature), washed with 1×HBSS and re-suspended at 1×10⁶ cells/ml inRPMI medium without serum. 1×10⁶ cells were incubated for 5 minutes withan exact antisense concentration (between 0 and 20 microM) in a sterilemicrotube. Each reaction was then transferred in 12 well plates andincubated at 37° C. for 5 hours. RPMI/FBS 20% was added to a finalconcentration of 10% FBS and cells were incubated at 37° C. overnight.Cells were harvested by centrifugation (5 minutes, 1500 RPM, at roomtemperature) and washed with 1× Hans Balanced Salt Solution (HBSS).

A549 Cell Line (Direct Transfection):

Cells were trypsinized, re-suspended in DMEM-F12 medium (10⁶ cells/well)and incubated at 37° C. overnight in serum deprivation conditions. Thenext day, adherent cells were incubated at 37° C. for 5 hours withantisense at different concentrations (0, 5, 10, 15 or 20 microM)directly in wells. DMEM-F12/FBS 20% was added to a final concentrationof 10% FBS and cells were incubated at 37° C. overnight. Cells wereharvested by centrifugation (5 minutes, 1500 RPM, at room temperature)and washed with 1×HBSS.

Escort II Reagent Mediated A549 Cell Line Transfection

A549 cells were seeded over night at 3×10⁵ cells/mL in 12 well plates at37° C., 5% CO₂. The day after, cells were washed twice with 1×HBSS andcovered with 500 microL serum and antibiotics free DMEM-F12.Transfection complex consisted of 1 microg of antisense and 2.5 microLEscort II reagent. The DNA-Escort II complex was prepared and added tothe cells according to recommendations of the manufacturer.

BSMC and NHBE Primary Cells:

The cells were trypsinized (trypsin 0.025%, EDTA 0.01%) for 3-5 minutesat 37° C., the reaction was stopped with culture medium, the cellscounted resuspended at 5×10⁵ cells/mL in 12 well plates and incubated at37° C. (overnight). The next day, cells were washed, the medium changedand replaced with culture medium without serum. Antisense was added atdifferent concentrations (between 0 and 20 microM) directly in thewells. The mixture was incubated at 37° C. for 5 hours. The serum wasadded to a final concentration of 5%, and the culture incubated foranother 48 hours. The cells were washed with HBSS 3 times, harvested andRNA was extracted.

PBMC

2×10⁶ cells/mL/well are incubated with antisense concentrations rangingfrom 0 to 5 microM in RPMI 1640 cell culture medium supplemented with 5%heat inactivated FBS, Penicillin 100 U/mL, Streptomycin 100 microg/mLand 10 microg/mL PHA for 16 to 20 hrs. After that cells are recoveredand washed before RNA preparation.

RNA Extraction

RNA is extracted from cell pellets according to RNAeasy mini Kitprotocol using the QiaVac 24 manifold from Qiagen and treated withDNase-I according to Fermentas procedures. RNA is quantified using theRiboGreen reagent according to the manufacturer protocol(Invitrogen-Molecular probes).

Reverse Transcription (RT)

Preparation of First-Strand cDNA was Performed Using the SuperscriptFirst-Strand Synthesis System for RT-PCR kit, in a total reaction volumeof 20 microL. 1 microg of RNA was first denatured at 65° C. for 5minutes, with 0.5 mM of each dNTPs, 0.5 microg of oligo (dT)₁₂₋₁₈ andchilled on ice for at least 1 min. The mixture was incubated at 42° C.for 2 minutes and a second pre-mix containing 1× First-Strand Buffer, 10mM DTT, 40 units of RNaseOUT and 40 units of SuperScript II RT wasadded. Reactions were incubated at 42° C. for 10 minutes, at 50° C. for1 hour and inactivated by heating at 70° C. for 15 minutes.

Polymerase Chain Reaction (PCR)

PCR was performed with optimized quantity of cDNA (10 to 200 nanogdepending on the gene target) in 1×PCR buffer (10×: Tris-HCl, KCl,(NH₄)₂SO4, 15 mM MgCl12; pH8.7)) in a total reaction volume of 50microL, 0.2 mM of each dNTPs, 8.5 pmol of each PCR primer and 2.5 unitsof Taq DNA Polymerase. The mixture was heated at 94° C. for 5 minutes,followed by 30 to 35 cycles (depending on targets), each consisting ofincubation for 1 minute at 94° C., 45 seconds at 60° C. and 45 secondsat 72° C. Supplemental elongation was performed at 72° C. for 10minutes. PCR products were analyzed by 1.5% agarose gel electrophoresisin the presence of ethidium bromide. PCR primer sequences used for eachgene and expected sizes of PCR amplification products are described inTables 2a-b. Quantification of PCR products was performed using theTotal Lab software (Background subtraction with Rolling Ball; Ultra LumInc., Model UC4800).

Real-Time PCR

PCR reaction mixtures are prepared with 3 microL of cDNA reaction in atotal volume of 20 microL in presence of 0.5 mM of each PCR primer and 4microL of LC FastStart DNA Master SYBR Green 1 PLUS.

Step 1 (Denaturation): 95° C., 10 min (slope 20° C./sec)Step 2 (Cycles×40): 95° C., (slope 20° C./sec); 57° C., 5 sec. (slope20° C./sec) (Except for PDE4D Tm=59° C.); 72° C., 10 sec. (slope 20°C./sec)Step 3 (Melting curve): 95° C., (slope 20° C./sec); 70° C., 30 sec.(slope 20° C./sec); 95° C., 0 sec. (slope 0.1° C./sec)Step 4 (Cool): 40° C., 30 sec (slope 20° C./sec)PCR primer sequences used for each gene are described in Tables 2c-d.Quantification of PCR products was performed using the RelQuant program(Roche).

Human TNF-Alpha ELISA

PBMC are transfected with antisense(s) (0.05 microM each) as describedbefore without PHA stimulation for 16-18 hrs. After that cells arestimulated with PHA (10 microg/mL) for 6 hrs. Supernatants are thereforecollected, centrifuged 5 min at 2000 rpm, stored at −80° C. until usedfor ELISA as described by the manufacturer.

Animals.

All animal protocols were approved by the Mispro Biotech Services Inc.Animal Care Committee and conform to the Canadian Council of Animal Careguidelines regarding animal experimentation. C57BL/6 and AKR/J mice(20-25 g) (Charles River Canada, Lasalle QC, Canada) were housed atMispro Biotech Services Inc. animal facility (Montreal QC, Canada).

In Vivo Antisense Targets Validation and Multiple Gene Cross Knock Down

C57BL/6 mice were lightly anesthetized by i.p. injection ofketamine/xylazine. For groups receiving the antisense oligonucleotides,mice were pre-treated by nasal instillation of the antisenseoligonucleotide solution (50 mg/mouse/antisense) 16-20 hrs. before lungsharvesting. Control mice received an identical amount of controlantisense oligonucleotide in sterile PBS.

Lipopolysaccharide(LPS)-Induced Lung Inflammation Model.

C57BL/6 mice were lightly anesthetized by i.p. injection ofketamine/xylazine and acute lung inflammation was induced by nasalinstillation of 10 mg of LPS from E. coli serotype 0111:B4(Sigma-Aldrich, Oakville ON, Canada). Control mice receive a similarvolume of sterile PBS. For groups receiving the antisenseoligonucleotides, mice were pre-treated by nasal instillation of theantisense oligonucleotide solution (200 mg/mouse) 12 h before LPSexposure. Control mice received an identical amount of control antisenseoligonucleotide in sterile PBS. Bronchoalveolar lavage and lungharvesting were performed 6 h after LPS instillation.

μμ

Cigarette Smoke-Induced Lung Inflammation Model.

AKR/J mice were exposed to the whole smoke from eight 1R3F researchcigarettes (University of Kentucky) using a smoking apparatus (SCIREQScientific Respiratory Equipment Inc., Montreal Canada). Control micewere sham smoked. For groups receiving the antisense oligonucleotides,mice were lightly anesthetized and were pre-treated by nasalinstillation of the antisense oligonucleotide (200 mg/mouse) solution 3h before and 24 h after cigarette smoke-exposure. Control mice receivedan identical amount of control antisense oligonucleotide in sterile PBS.Bronchoalveolar lavage and lung harvesting were performed 48 h aftercigarette smoke exposure.

Bronchoalveolar Lavage (BAL) and Differential Cell Count.

Mice were anesthetized by i.p. injection of ketamine/xylazine. Atracheotomy was performed and the lungs were washed 4 times with 0.5 mlof ice-cold PBS. Total cells were counted and then centrifuged onto acytospin slide. The differential cell count was assessed after cytospinslides were stained with Wright-Giemsa. At least 200 cells were countedunder oil immersion microscopy. BAL fluids were stored at −80° C. forsubsequent TNF-alpha measurements.

Mouse TNF-Alpha ELISA.

Levels of TNF-alpha in BAL fluid were measured using a murine TNF-alphaELISA kit according to the manufacturer recommendations.

Reverse Transcription (RT) and Real-Time PCR.

Mice Lungs were homogenized using a polytron PT 1200 (BrinkmannInstruments) and total RNA was extracted using the Qiagen RNAeasy minikit (Qiagen, Mississauga ON, Canada) followed by DNase I digestion.Total RNA was quantified using the Ribogreen Fluorescent Assay(Invitrogen Corporation, Burlington ON, Canada). cDNA was prepared from1 microg RNA using the First-Strand cDNA Synthesis Using SuperScript™ IIRT kit (Invitrogen Corporation, Burlington ON, Canada). Real-time PCRreactions were prepared with 50 nanog cDNA using the LC FastStart DNAMaster SYBR Green 1 PLUS (Roche Diagnostics, Laval QC, Canada) and runin a LightCycler (Roche Diagnostics, Laval QC, Canada). Primer sets weredesigned by TibMolbiol (Adelphia, N.J.) and GSP purified (see Table 2d).PBGD or HPRT were used as a reference gene. Each real-time PCR runincluded a calibrator cDNA (prepared from pooled RNA from mouse lungtissue) and gene/PBGD or gene/HPRT ratios were normalized using the Cpvalues of the calibrator. In addition, standard curves for each gene,for PBGD and for HPRT were done using a calibrator cDNA, and coefficientfiles were created to include correction for the differences inamplification efficiency between target and reference genes.

EXAMPLES Example 1

This example relates to PDE Isotype cell and tissue distribution.Inflammatory respiratory diseases are characterized by an interactionbetween inflammatory cells (macrophages, lymphocytes, neutrophils andeosinophils) and tissue cells (mostly epithelial cells and smooth musclecells). mRNA expression of the isotypes of PDE 3 (PDE3A and B), PDE 7(PDE7A and B) and of PDE 4 (PDE4A, B, C, and D) in different cells andtissue was assessed. A summary of the primers used for every PDE isotypecan be found in Table 2a. The cells that were chosen were A549(epithelial cell from an adenocarcinoma of the lung), and NCI-H292(mucoepidermoid pulmonary carcinoma). Expression in primary human cellswas assessed in BSMC (Human bronchial smooth muscle cells) and PBMC(Peripheral blood mononuclear cells). Normal human lung was also used tocharacterize the expression of PDEs. Finally, normal human brain wasincluded as a control, since most PDEs are expressed in this tissue. Ascan be seen in FIG. 1 different cells and tissues express differentisotypes of PDEs except for PDE 7A and PDE4B that were expressed inevery cell and tissue assessed under the conditions that were specifiedabove. PDE4C mRNA was found less abundant in cells and tissues used inthese experiments. PDE4A was found in the lung and brain while PDE4D wasconfined to the lung and airway cells (A549 and NCI—H292). Since PDE7Awas present in all the cells tested and has recently been suggested tobe important in diseases mediated by lymphocytes, we also assessed thesub-isotypes of PDE7A. As can be seen in FIG. 2, PDE7A1 appears to bethe isotype that is the most expressed in all the cells that weretested.

The PCRs were repeated several times and the presence or absence ofexpression of an isotype of the PDE enzymes in each cell line, primarycells or tissues is presented in Table 3a and b. It should be noted thatPDE3A was not found in the PBMC and several cell lines except A549.Moreover, messengers corresponding to PDE7B and PDE4C were rarelydetected in cells and tissues used here.

Example 2

This example relates to effective antisense oligonucleotides againstPDE7A. Several antisense oligonucleotides against PDE7A mRNA weredesigned and assessed for their efficacy in the cells that expressPDE7A. Table 1a includes a list of the sequences that were used. All theexperiments were performed with increasing concentrations of theoligonucleotides and efficacy was assessed by comparing the ratio ofPDE7A mRNA density over GAPDH density in the treated cells to that ofthe untreated cells.

It is to be noted in FIG. 3 that Top-1702 (SEQ. ID NO.: 1), Top-1706(SEQ. ID NO.: 3), Top-1703 (SEQ. ID NO.: 2), Top-1701, and Top-1707,were found effective at inhibiting PDE7A mRNA expression in Eol-1 cells.In addition Top-1702 (SEQ. ID NO.: 1), Top-1703 (SEQ. ID NO.: 2),Top-1706 (SEQ. ID NO.: 3), and Top-1707, inhibited PDE7A mRNA in U937cells. In FIG. 4, several PDE7A antisense oligonucleotides displayedmore than 60% inhibition of PDE7A mRNA in Jurkat cells (Top-1702 (SEQ.ID NO.: 1), Top-1703 (SEQ. ID NO.: 2), and Top-1706 (SEQ. ID NO.: 3)) atconcentrations of 10 to 20 micromolar. The oligonucleotides Top-1707 andTop-1726, were practically ineffective under the conditions studied. InHut-78 cells the oligonucleotide Top-1703 (SEQ. ID NO.: 2) retained itsefficacy at a concentration of 10 micromolar while Top-1706 (SEQ. IDNO.: 3) was less effective and Top-1702 (SEQ. ID NO.: 1) was not aseffective. In BSMC and A549 cells, Top-1702 (SEQ. ID NO.: 1), Top-1703(SEQ. ID NO.: 2) and Top-1706 (BSMC only shown, SEQ. ID NO.: 3) were alleffective at downregulating mRNA expression of PDE7A as shown in FIG. 5.Moreover, these PDE7A antisenses were assessed in human PBMC. Theresults showed that among antisenses, Top-1706 (SEQ. ID NO.: 3) waseffective at inhibiting it's specific messenger at 40%, Table 4a. Theresults reported here were target and antisense oligonucleotide primarysequence specific and were not due to an indirect or non-specificeffect.

The overall results suggest that the antisense strategy towards PDE7A ofthe present invention is successful in providing significant inhibitionof targeted mRNA. Among the designed oligos, Top-1703 (SEQ. ID NO.: 2),Top-1706 (SEQ. ID NO.: 3), and Top-1702 (SEQ. ID NO.: 1), seem the mosteffective at inhibiting mRNA expression for PDE7A in all the cell linesand primary cells studied. These results suggest that these antisenseoligonucleotides could be used in the therapeutic based strategy of thepresent invention.

Example 3

This example relates to effective antisense oligonucleotides againstPDE3A and PDE3B. A set of antisense oligonucleotides were designed andtested for their ability to decrease mRNA in several cell types. Table1b-c includes a list of the sequences that were used.

Several antisense oligonucleotides were found to be effective atreducing PDE3A mRNA level in A549 cells and/or in U937 cells includingTop-1301 (SEQ. ID NO.: 10), Top-1302 (SEQ. ID NO.: 11), Top-1303 (SEQ.ID NO.: 12), Top-1307 (SEQ. ID NO.: 13), Top-1308 (SEQ. ID NO.: 14), TOP1309 (SEQ. ID NO.: 15), Top-1311 (SEQ. ID NO.: 16) and Top-1312(although less effective) as shown in FIG. 6, Table 4a (above) and 4b(below). In primary bronchial smooth muscle cells the same antisenseoligonucleotides were also effective at decreasing PDE3A mRNA expressionas shown in FIG. 7.

Experiments performed with PBMC using the PDE3B showed that severalantisenses were effective at blocking specifically the PDE3B mRNA, amongthese antisenses Top-1357 (SEQ. ID NO.: 18) showed 75% inhibition whileTop-1351 showed 4%, as illustrated in Table 4a

Taken together these results suggest that the antisense strategy of thepresent invention towards PDE3A and/or PDE3B inhibition is effective.Several other antisense oligonucleotides, Top-1304, Top-1305, Top-1313,Top-1351 did not provide significant mRNA inhibition, suggesting thatthe antisense blocking efficiency reported here was target and antisenseoligonucleotide primary sequence specific but was not due to an indirectantisense effect.

Example 4

This example relates to effective antisense oligonucleotides againstPDE4. Several antisense oligonucleotides against PDE4 mRNA were designedand assessed for their efficacy in the cells that express PDE4. Table1d-f include a list of the sequences that were employed. All theexperiments were performed with increasing concentrations of theoligonucleotides and efficacy was assessed by real time PCR comparingthe ratio of PDE4A, -B, -D, mRNA density over HPRT (HYPOXANTHINEPHOSPHORIBOSYLTRANSFERASE, as an internal control) density in thetreated cells to that of the untreated cells (Table 2c and d).

Several antisense oligonucleotides were found to be effective atreducing PDE4 mRNA level in PBMC including Top-1411 (PDE4A, SEQ. ID NO.:21), Top-1437 (PDE4B, SEQ. ID NO.: 28) and Top-1498 (PDE4D, SEQ. ID NO.:36), at 43, 53 and 56%, respectively, Table 4a.

Taken together these results suggest that the antisense strategy of thepresent invention towards PDE4 inhibition is effective. Several otherantisense oligonucleotides, Top-1406 (PDE4A), Top-1430 (PDE4B), Top-1495(PDE4D), did not provide significant mRNA inhibition, suggesting thatthe antisense blocking efficiency reported here was target and antisenseoligonucleotide primary sequence specific but was not due to an indirectantisense effect.

Example 5

This example relates to the effect of inhibition of a single isotype ofPDE on mRNA production of a different PDE. The experiments wereconducted in A549 cells and in PBMC. As shown in Table 4b, severalantisenses were found not only to inhibit their specific targets butwere able to down regulate mRNA corresponding to other PDEs. PDE3Aantisense Top-1308 (SEQ. ID NO.: 14) not only provided inhibition ofit's specific mRNA in A549 cell line (60%), but also downregulated theexpression of PDE3B (53%), PDE4A (63%), PDE4D (26%) and PDE7A (44%). Thesame experiments were done in PBMC and the results showed that, PDE3Bantisense Top-1357 (SEQ. ID NO.: 18) has the capacity to inhibitspecifically it's target (62%) and was also able to down regulate themessengers corresponding to PDE4A, -4B, -4D and 7A by 76, 43, 65 and 38%respectively, Table 4b. This multiple gene knock down process was alsofound with antisenses directed against PDE4A Top-1413 (SEQ. ID NO.: 23),against PDE4B Top-1437 (SEQ. ID NO.: 28), against PDE4D Top-1490 (SEQ.ID NO.: 34), against PDE7A Top-1706 (SEQ. ID NO.: 3) were used in PBMC,Table 4b. The observed inhibition was specific for the followingreasons: All Top-antisense oligonucleotides employed in this study didnot have any homology with the other PDEs nucleotide sequences studied.In addition, the effective antisenses used here did not affect all thePDEs studied, for example Top-1308 (SEQ. ID NO.: 14) which was potentagainst PDE3B, -4A and 7A was at lesser extent effective against -4D buthad no effect on PDE4B. Moreover, several other antisenses used in thisstudy were neither found active against their specific target noragainst the other PDEs, (Top-1312, Top-1404), Table 4b. These resultsshow that inhibition of a PDE isotype mRNA with antisenseoligonucleotides affects the production of other PDEs and accounts forthe single oligonucleotide induced multiple gene knock down process asdescribed herein.

Example 6

This example relates to the effect of inhibition of a single isotype ofPDE on cytokine and enzyme mRNA production. Cytokines and chemokines aremediators that are important in cell activation and recruitment. Amongstthe mediators that have been shown to be increased and/or play a role inthe pathophysiology of inflammatory respiratory diseases are interleukin(IL)-6, IL-7, IL-8, IL-15 and TNF-alpha. Several enzymes are directly orindirectly involved in tissue destruction and/or repair. Amongst theseenzymes are matrix metalloproteinases, MMPs (MMP-1, -2, -3, -12), atissue specific inhibitor of MMPs 1 (TIMP-1) and cyclooxygenase cox-1.It has been shown in several inflammatory respiratory diseases thatthere is an increase in the ratio of MMPs to their inhibitors or TIMPs.The effects of inhibition of PDE3A or of PDE7A mRNA on mediator andenzyme mRNA production by human primary smooth muscle bronchial cellswas assessed.

As shown in FIG. 8, the antisense oligonucleotides that were effectiveat decreasing PDE3A (Top-1301 (SEQ. ID NO.: 10), Top-1303 (SEQ. ID NO.:12), Top-1308 (SEQ. ID NO.: 14), Top-1311 (SEQ. ID NO.: 16)) or PDE7A(Top-1702 (SEQ. ID NO.: 1), Top-1703 (SEQ. ID NO.: 2), Top-1706 (SEQ. IDNO.: 3) mRNA production also decreased mRNA for IL-6, IL-7, IL-15,TNF-alpha and to a lesser degree mRNA for IL-8. The antisenseoligonucleotides also had an inhibitory effect on mRNA for MMP12 but, atthis concentration, there were no effects on MMP-1, MMP-2, MMP-3, TIMP-1and cox-1 mRNA.

The effect of single PDE specific antisenses on other PDEs is given inTable 4b. Several specific PDE antisenses ((Top-1308 (SEQ. ID NO.: 14);Top-1357 (SEQ. ID NO.: 18); Top-1413 (SEQ. ID NO.: 23); Top-1437 (SEQ.ID NO.: 28); Top-1490 (SEQ. ID NO.: 34); Top-1706 (SEQ. ID NO.: 3)) notonly have efficacy in down regulating their specific targets but havealso inhibitory effects on other phosphodiesterases.

Here again, the observed inhibition was specific since all Top-antisenseoligonucleotides employed in this study did not have any similarity withthe cytokine or enzyme nucleotide sequences studied. In addition thelevel of Top-antisense decrease in mRNA production was sequence andcytokine specific. These results show that inhibition of a PDE isotypemRNA with antisense oligonucleotides affects mediator and enzymeproduction.

Example 7

This example relates to the effect of combination of isotype specificantisense oligonucleotides on PDE3A and PDE7A gene expression. Theeffects of combining oligonucleotides on PDE3A and PDE7A mRNA expressionin U937 cells that express both isotypes of PDEs was assessed. For this,each oligonucleotide was employed separately at a concentration that waspractically ineffective in this cell line under the conditions studied(10 μmolar, as shown in FIG. 9). Certain combinations of PDE3A and PDE7Aantisense oligonucleotides were significantly much more effective atdecreasing both PDE3A and PDE7A mRNA expression than either antisenseoligonucleotide alone. The combination of Top-1703 (SEQ. ID NO.: 2) andTop-1301 (SEQ. ID NO.: 10) or Top-1706 (SEQ. ID NO.: 3) and Top-1303(SEQ. ID NO.: 12) exhibited a strong synergistic inhibition of bothPDE3A and PDE7A mRNA. In addition, the effects reported here are targetand antisense primary sequence specific, since several other TOP-1700and TOP-1300 oligonucleotide combinations did not have a significantmRNA inhibitory effect.

The effective PDE oligonucleotide antisense compounds for use in thepresent combinations are described herein as those capable of inhibitingexpression of a target PDE gene, which when combined with anotheroligonucleotide compound, both at a concentration that provides lessthan 20% inhibition of their target PDE gene, causes at least one of thetarget PDE genes to be inhibited by more than 20% and at least doublethe amount of inhibition that is exhibited by one oligonucleotidecompound alone. The combination may exhibit inhibition of both PDEnucleic acid targets as well as nucleic acid encoding other PDEs andinflammatory mediators.

These results show a specific, multiple gene knock-down effect ofcombining two antisense oligonucleotides derived from the nucleotidesequences of two different target genes, such as PDE7A and PDE3A.Although the oligonucleotides were administered at concentrations thathad no effect on their target genes when employed alone, thecombinations induced significant inhibition on the target genes.

Example 8

This example relates to the effect of the combination of PDE3A and PDE7Aantisense oligonucleotides on mediator and enzyme mRNA expression. Anexample of the effects of multiple gene knock-down on the expression ofmRNA for cytokine, chemokine and enzymatic genes is illustrated in FIG.10. Results were obtained in the U937 monocytic cell line. It is to benoted that at the concentration studied each antisense oligonucleotidealone has a small inhibitory effect on IL-10 and MMP-8 mRNA expression.The antisense oligonucleotides when employed alone have no inhibitoryeffect on PDE4B, MMP-1, MMP-2 and TIMP-1 mRNA expression. Thecombination of 2 antisense oligonucleotides (Top-1311 (SEQ. ID NO.:16)+Top-1703 (SEQ. ID NO.: 2) or Top-1307 (SEQ. ID NO.: 13)+Top-1703(SEQ. ID NO.: 2)) has the same effect as each antisense oligonucleotideemployed alone (mild inhibitory effects on IL-10 and MMP-8 mRNA with noeffects on PDE4B, MMP-1, MMP-2 and TIMP-1 mRNA expression. However, thecombination of 2 antisense oligonucleotides (Top-1301 (SEQ. ID NO.:10)++Top-1703 (SEQ. ID NO.: 2) or Top-1303 (SEQ. ID NO.: 12)+Top-1706(SEQ. ID NO.: 3) had a synergistic inhibitory effect as shown by thesignificant decrease in not only IL-10 and MMP-8 mRNA expression butalso PDE4B, MMP-1 and MMP-2 mRNA expression without affecting TIMP-1mRNA expression. The multiple gene knock down that is found by employingthe last 2 combinations of antisense oligonucleotides is selective sinceTIMP-1, a gene that inhibits the enzymatic activity of several MMPs wasunaffected and is specific since the housekeeping gene GAPDH was notaffected by the combination. These results are in concurrence with thosepresented in example 5 and show the potential for the multiple geneknock down to have a broader anti-inflammatory effect.

Example 9

This example employs a functional assay (ie inhibition of TNF-alphaproduction) to illustrate the multiple gene knock down effect ofcombining 2 selected oligonucleotides against PDE isotypes in humanPBMC.

Cytokines play a critical role in the orchestration of chronicinflammation in all diseases, including asthma and COPD. TNF-alpha isone of the most important pro-inflammatory cytokines that is expressedin several inflammatory diseases. TNF-alpha levels are markedlyincreased in induced sputum of patients with COPD (Keatings et al., Am.J. Respir. Crit. Care Med. 1996; 153; 530-534.). Furthermore, there isevidence that COPD patients with weight loss show increased release ofTNF-alpha from circulating cells and that TNF-alpha may induce apoptosisof skeletal muscle cells, resulting in the characteristic muscle wastingand cachexia seen in some patients with severe COPD (De Godoy et al.,Am. J. Respir. Crit. Care Med. 1996, 153, 633-637.).

PDE4 inhibitors caused a decrease in the release of cytokines andchemokines from inflammatory cells via an increase in intracellularcyclic AMP (Torphy, Am. J. Respir. Crit. Care Med. 1998, 157, 351-370).In contrast to corticosteroids, PDE4 inhibitors have a potent inhibitoryeffect on neutrophils (Au et al., Br. J. Pharmacol. 1998, 123,1260-1266.) indicating that they may be useful anti-inflammatorytreatments for COPD. There is preliminary evidence that a PDE4 inhibitorCilomilast improves lung function and symptoms in patients with COPD andthis may be due to cytokine inhibition (Compton et al., Lancet. 2001,358, 265-270). Here it is shown that a combination of several selectedantisense oligonucleotides, not only inhibited their specific PDEtargets but also caused multiple gene knock-down, including a decreasein the production of the pro-inflammatory cytokine TNF-alphã FIG. 11shows that when antisense oligonucleotides (Top-1360 (PDE3B, SEQ. IDNO.: 19); Top-1413 (PDE4A, SEQ. ID NO.: 23); Top-1437 (PDE4B, SEQ. IDNO.: 28); Top-1498 (PDE4D, SEQ. ID NO.: 36)) were used individually theyhave a small, minimal effect on TNF-alpha release from PHA-activatedPBMC at the concentration tested. However, when combinations wereassessed, they provide a more than additive effect on TNF-alpha releasefrom PBMC. The observed effect on TNF-alpha release was specific sincethe use of mismatch antisenses individually or in combination with otherantisenses had no effect on TNF-alpha release from stimulated PBMC.

Because TNF-alpha, is considered as a common denominator in inflammatoryand chronic respiratory diseases, this invention provides a broadtherapeutic application for treating several inflammatory diseases.

Example 10

This example shows that it is possible to decrease expression of mRNAfor PDE4A, PDE4B and PDE7A by administering an antisense oligonucleotidedirected against a PDE isotype in vivo. In this experiment, eachantisense oligonucleotide (Table 5) was administered by nasalinstillation (50 mg) and lungs collected 16 h later. The expression oftarget mRNA was assessed by real-time PCR on the cDNA prepared fromwhole lung. Table 5 lists several antisense oligonucleotides specific toeach murine target mRNA for which the inhibitory activity measured wasgreater than 25%. A control antisense oligonucleotide that was includedin each experiment showed no inhibitory activity of the different mRNAtargets suggesting that the antisense activity observed was target andantisense oligonucleotide sequence specific. Therefore, inhibition ofthe expression of several PDE mRNAs can be achieved in the lung of miceby simple nasal instillation of active antisense oligonucleotides.

Example 11

This example demonstrates that a combination of 2 antisenseoligonucleotides can be employed to induce multiple gene knock down invivo. FIG. 12A shows that at the time point measure TOP 2437, a murineantisense oligonucleotide directed against PDE4B has no significantinhibitory effect on PDE4B mRNA expression on its own when used at lowconcentration (50 mg/mouse). However, combining Top 2437 (an antisenseoligonucleotide directed against PDE4B) with Top 2713 (an antisenseoligonucleotide directed against PDE7A) leads to a more than 40%inhibition of PDE4B in mice lungs. FIG. 12B shows that although TOP2437and TOP2713 alone had little inhibitory effect on PDE7A mRNA expressionwhen used at low concentrations, the combination of both antisenseoligonucleotides inhibited PDE7A mRNA expression by 83%. Results showthat a combination of two antisenses targeting two different PDEisozymes resulted in higher level of inhibition of each target gene thansingle antisense treatment, e.g. a more than additive level ofinhibition.

FIG. 12C shows that a combination of antisense oligonucleotides directedagainst PDE4B and PDE7A will also decrease PDE4A mRNA expression inmurine lungs. Results showed that even though each antisenseoligonucleotide had no effect on its own, a high synergistic level ofinhibition of PDE4A mRNA expression was achieved following thecombination treatment.

These results demonstrate not only that a more than additive inhibitionof targeted PDE isotypes can be obtained by combining 2 antisenseoligonucleotides directed against these isotypes, but that additionalinhibition of other PDE isotypes can also be achieved by treatment witha combination of two antisenses that were not directed against a givenisotype.

Example 12

This example shows that an antisense oligonucleotide against a specificPDE isotype can partially inhibit the acute lung inflammation induced inmice by LPS (FIG. 13). PDE4B was targeted in this example because it waspreviously shown to be of importance in the LPS induced inflammatory (Maet al., Mol. Pharmacol., 1999, 55, 50-57.; Wang et al., Molec.Pharmacol., 1999, 56, 170-174.). In addition, our previous resultsconfirmed that PDE4B expression was up-regulated by LPS treatment inmice (data not shown).

To demonstrate that expression of target mRNAs could be inhibited inLPS-exposed mice, mice were pre-treated by nasal instillation of 200 mgof TOP2430 (specific to PDE4B) or control antisense oligonucleotide 12 hprior to LPS exposure. As shown in FIG. 13A, PDE4B expression wasreduced by 86% following TOP2430 pre-treatment. FIG. 13A also shows thatlung PDE4A, 4D and 7A mRNA expression was also partially inhibited byemploying an antisense oligonucleotide against PDE4B. In contrast, PDE3Bexpression was stimulated following the inhibition of PDE4B by 117%.This example demonstrates in another way the concept of multiple geneknock down in vivo.

FIG. 13B shows the effect of PDE4B inhibition on the LPS-induced acutelung inflammation. Results show that inhibition of PDE4B by TOP2430significantly reduced the cellular influx (40% reduction, p<0.013) inthe lungs of mice exposed to LPS when compared to control mice. Intreated mice, the reduction of total cells was associated with asignificant decrease in neutrophils (40% reduction, p<0.016) and inmacrophages (50% reduction, p<0.002) that were present in the lunglavage. Moreover, pre-treatment with TOP2430 caused a significantdecrease (26%) in LPS-induced TNF-alpha concentration in lung lavagefluid (FIG. 13C).

Results show that specific inhibition of target PDE mRNA can be achievedby topical delivery of antisense oligonucleotides in mice. Moreover,inhibition of PDE4B expression in LPS-exposed mice has a significanteffect on the inflammatory response with decreased cellular influx,neutrophil and macrophage migration to the lung and a decrease inTNF-alpha release. The mechanism of this effect may be related to themultiple gene knock down that occurs when an antisense oligonucleotidedirected against PDE4B results also in the down-regulation of severalother PDEs.

Example 13

This example relates to the efficacy of antisense oligonucleotidestargeting at specific PDEB4 mRNAs to inhibit acute lung inflammationinduced by cigarette smoke exposure (FIG. 14).

FIG. 14 shows the effect of having AKR/J mice breathe in cigarettesmoke. Smoke produced a significant increase in bronchoalveolar lavagecells (neutrophils and macrophages) by 24 h after exposure thatpersisted for at least 48 hours (FIG. 14A). In conjunction with theinflux of inflammatory cells, smoke exposure induced an increasedexpression of TNF-alpha in the lung tissue (3.5-fold increase, FIG.14B). It has been shown that macrophages, neutrophils and TNF-alpha playimportant roles in smoke-induced lung inflammation and progression tosubsequent emphysema (Churg et al., Am. J. Respir. Crit. Care Med.,2002, 166, 849-854.; Am. J. Respir. Cell Mol. Biol., 2002, 27, 368-374).

FIG. 14C shows the effect of PDE4B inhibition on smoke-induced increaseof TNF-alpha mRNA expression. Results showed that inhibition of PDE4B by20% with TOP2430 reduced the expression of TNF-alpha in lung tissue by21% when compared to mice that were pre-treated with a control antisenseoligonucleotide.

Taken altogether, these results show that specific inhibition of PDE4BmRNA expression can be obtained in smoke-exposed mice and that it has acomparable effect on TNF-alpha mRNA expression in whole lung tissue.

TABLE 1a Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1702GAACCTCTTCTTTCTGATTC PDE7A NM_002603 1 TOP-1703 GGTGAGAACCTCTTCTTTCTPDE7A NM_002603 2 TOP-1706 TTCCCACGTCCGACATG PDE7A NM_002603 3 TOP-1714GCTTAGGTTCCTTTAAGT PDE7A NM_002603 4 TOP-1716 TCATGAGTGGCAGCTGC PDE7ANM_002603 5 TOP-1718 CCATTTGTTGCCTGCTTTC PDE7A NM_002603 6 TOP-1719GTCTCCATTTGTTGCCTGC PDE7A NM_002603 7 TOP-1720 TTTCACTCCACTGCTTGCT PDE7ANM_002603 8 TOP-1722 CTGTGGTAATCTTCTTGAA PDE7A NM_002603 9

TABLE 1b Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1301GACTCGTGCAGCGTCGCC PDE3A NM_000921 10 TOP-1302 GTTCCTGACTCGTGCAG PDE3ANM_000921 11 TOP-1303 GGCTTGTTCCTGACTCGTGC PDE3A NM_000921 12 TOP-1307GCTCCGGAGCGGCTGCAGC PDE3A NM_000921 13 TOP-1308 GGCCAGCAGCGCGACAGCCAGPDE3A NM_000921 14 TOP-1309 GCGGACCAGCCTCACC PDE3A NM_000921 15 TOP-1311CGGCCAAGCAGCTGAGCACC PDE3A NM_000921 16

TABLE 1c Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1353GGCGGCTGCAGGGACCGC PDE3B NM_000922 17 TOP-1357 CCGCAGCTCCACGTTGCAG PDE3BNM_000922 18 TOP-1360 TCAGCAGCGTCCGCAGCCAG PDE3B NM_000922 19

TABLE 1d Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1403CGGAGGCTGGCCAGCACCTG PDE4A NM_006202 20 TOP-1411 TCTTGCGTAGGCTCTGC PDE4ANM_006202 21 TOP-1412 CACTTTCTTGGTCTCCACC PDE4A NM_006202 22 TOP-1413GCCACGCTCGCGCTCTC PDE4A NM_006202 23 TOP-1505 GTCCACTGGCGGTACAG PDE4ANM_006202 24 TOP-1510 GTCATAGTCGCTGTCTG PDE4A NM_006202 25 TOP-1512CCATGATGCGGTCTGTCCA PDE4A NM_006202 26

TABLE 1e Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1432GACCGGTAGGTCTGTATGGT PDE4B NM_002600 27 TOP-1437 GGAGGTCTCTTTCCTGG PDE4BNM_002600 28 TOP-1534 CGGTGCTACTGAAGGTG PDE4B2 NM_002600 29 TOP-1537CTGATTCCGGTGCTACT PDE4B2 NM_002600 30 TOP-1538 CTGATTCCGGTGCTACTG PDE4B2NM_002600 31 TOP-1539 CTGATTCCGGTGCTACTGA PDE4B2 NM_002600 32

TABLE 1f Antisense sequence Antisense identification Target AccessionSeq identification (5′-3′) gene number ID No. TOP-1489CCGGTCCGTCCACTGGCG PDE4D NM_006203 33 TOP-1490 CGTTCCCTCTCTCGGTCTCCPDE4D NM_006203 34 TOP-1497 CTGCCTCCTCTTCAACCTG PDE4D NM_006203 35TOP-1498 CTTCAGGCTGGCTTTCCTC PDE4D NM_006203 36

TABLE 2a PDE SENSE PRIMER ANTISENSE PRIMER PRODUCT SEQ ID ISOFORM(5′-3′) (5′-3′) SIZE (bp) NOS PDE3A CCTATTCCAGGCCTCTCAACGCCAACCGTTGCTCCTCTTC 940 37/38 PDE3B GGAAGGATTCTCAGTCAGGCAGTGAGGTGGTGCATTAG 1085 39/40 PDE7A CCTTACCATAACGCAGTCCGAGCACCTATCTGTGTCTC 284 41/42 PDE7A1 CCAGCCTCTGCTGCCTCTGGCGCTTACCTACCAACCTTCC 665 43/44 PDE7A2 CTGGTGTCTGGCCTTGGTTCGCATACACTTGGCTTGTCC 348 45/46 PDE7A3 CGAAGACCAGCTTCTGCCACGGCCTCGTGCTTCTGGTGTGG 379 47/48 PDE7B CCACCTCGCACACAACAAGGGCAGCGAATGCTGAGGTGAC 305 49/50 PDE4A CAACGATGAGTCGGTGCTCGCACTGCCTCCAGCTCGGCCTCC 802 51/52 PDE4B CTGCACGCTGCTGATGTAGCCCTGAGCATCAGGCTGTACC 654 53/54 PDE4C GACGCCTCGGTGCTGGAGAACGATCTTGCTCTGGTACCAC 518 55/56 PDE4D GACATCGTACTTGCAACAGGTACCATTCACGATTGTCC 385 57/58

TABLE 2b SENSE PRIMER ANTISENSE PRIMER PRODUCT SEQ ID (5′-3′) (5′-3′)SIZE (bp) NOS IL-6^(a) CTTTTGGAGTTTGAGGTATACCTAGCTGCGCAGAATGAGATGAGTTGTC 236 59/60 IL-7^(b) TTTTATTCCGTGCTGCTCGCGCCCTAATCCGTTTTGACCA 429 61/62 IL-8^(a) ACATACTCCAAACCTTTCCACTCTTCAAAAACTTCTCCACAAC 168 63/64 IL-10^(b) AAAAGAAGGCATGCACAGCTCCAATAAGGTTTCTCAAGGGGCTGG 653 65/66 IL-15^(b) TGTCTTCATTTTGGGCTGTTTCATCCTCCAGTTCCTCACATTCTTTG 327 67/68 TNFalpha^(a) AACGGAGGCTGAACAATAGGCAGCAACCTTTATTTCTCGCCAC 206 69/70 MMP-1^(c) CGACTCTAGAAACACAAGAGCAAGAAAGGTTAGCTTACTGTCACACGCTT 786 71/72 MMP-2^(c) GTGCTGAAGGACACACTAAAGAAGATTGCCATCCTTCTCAAAGTTGTAGG 580 73/74 MMP-3^(c) AGATGCTGTTGATTCTGCTGTTGAGACAGCATCAAAGGACAAAGCAGGAT 515 75/76 MMP-8^(c) GCTGCTTATGAAGATTTTGACAGAGACAGCCACATTTGATTTTGCTTCAG 435 77/78 MMP-12 CCTTGCCATCTGGCATTGAAGGGTGATACGTTGGAGTAGGAAGTC 397 79/80 TIMP-1^(d) GGCCTTAGGGGATGCCGCGGCTATCTGGGACCGCA 403 81/82 Cox-1^(d) TGCCCAGCTCCTGGCCCGCCGCTTGTGCATCAACACAGGCGCCTCTTC 303 83/84 PBGD TGCAACGGCGGAAGAAAACGGCTCCGATGGTGAAGCC 313 85/86 GAPDH GGTAAAGTGGATATTGTTGCCAGTCTTCTGGGTGGCAGTG 448 87/88 ^(a)Gaede, K. I. Et al. (1999) J. Mol.Med. 77, 847-852 ^(b)Kebelmann-Betzing, C. et al. (2001) Cytokine. 13,39-50. ^(c)Ishii, Y. et al. (2003) Int. J. Cancer. 103, 161-168.

TABLE 2c Gene Primer name Primer sequence (5′-3′) DE3A hu/muPDE3A FCAACAGTGACAGCAGTGACATT (SEQ ID NO: 89) hu/muPDE3A RTTGAGTCCAGGTTATCCATGAC (SEQ ID NO: 90) PDE3B huPDE3B F1GATTCTTTGGGATTGGGACT (SEQ ID NO: 91) hu/muPDE3B R1 ATCTTTGGCCTACAGGAACC(SEQ ID NO: 92) PDE4A hu/muPDE4A F1 ATCAACACCAATTCGGAGCT (SEQ ID NO: 93)hu/muPDE4A R1 CCAGCACCATGTCGATGAC (SEQ ID NO: 94) PDE4B huPDE4B F1TGGCAGACCTGAAGACAATG (SEQ ID NO: 95) huPDE4B R2 AAATTCCTCCATGATGCGG (SEQID NO: 96) PDE4D hu/muPDE4D F1 CAGAATATGGTGCACTGTGC (SEQ ID NO: 97)hu/muPDE4D R1 AGTCTATGAAGCCCACCTGTG (SEQ ID NO: 98) PDE7A hu/muPDE7 F2TCAGGCCATGCACTGTTACT (SEQ ID NO: 99) huPDE7 R2 CCTGATTCTCTCAATAAGCCC(SEQ ID NO: 100) TNF-alpha TNFa-F AACGGAGGCTGAACAATAGGC (SEQ ID NO: 101)TNFa-R AGCAACCTTTATTTCTCGCCAC (SEQ ID NO: 102) IL-6 hu IL-6 FCTTTTGGAGTTTGAGGTATACCTAG (SEQ ID NO: 103) hu IL-6 RCGCAGAATGAGATGAGTTGTC (SEQ ID NO: 104) IL-10 hu IL-10 FTGCTGGAGGACTTTAAGGGTTAC (SEQ ID NO: 105) hu IL-10 RGTAGATGCCTTTCTCTTGGAGC (SEQ ID NO: 106) HPRT HPRT exon 3.4ATCAGACTGAAGAGCTTTGTAATGACCA (SEQ ID NO: 107) HPRT exon7TGGCTTATATCCAACACTTCGTG (SEQ ID NO: 108)

TABLE 2d Gene Primer name Primer sequence (5′-3′) DE3B muPDE3B F1ATCCTCTGGGACTGGGACTT (SEQ ID NO: 109) hu/muPDE3B R1 ATCTTTGGCCTACAGGAACC(SEQ ID NO: 110) PDE4A muPDE4A F2 GCGTCTCCAACCAGTTCCTA (SEQ ID NO: 111)hu/muPDE4A R1 CCAGCACCATGTCGATGAC (SEQ ID NO: 112) PDE4B muPDE4B F1AAGGTGACAAGCTCCGGT (SEQ ID NO: 113) hu/muPDE4B R1 TCTTTGTCTCCCTGCTGGA(SEQ ID NO: 114) PDE4D hu/muPDE4D F1 CAGAATATGGTGCACTGTGC (SEQ ID NO:115) hu/muPDE4D R1 AGTCTATGAAGCCCACCTGTG (SEQ ID NO: 116) PDE7Ahu/muPDE7 F1 CTCAGGCCATGCACTGTTAC (SEQ ID NO: 117) muPDE7 R2GGCAAGTGTGAGAACAAACC (SEQ ID NO: 118) TNF-alpha muTNF-alpha FTGCTCAGAGCTTTCAACAACTACTC (SEQ ID NO: 119) muTNF-alpha RGAGGCTCCAGTGAATTCGGA (SEQ ID NO: 120) IL-6 mu IL-6 FATGGATGCTACCAAACTGGAT (SEQ ID NO: 121) mu IL-6 R GCCACTCCTTCTGTGACTCC(SEQ ID NO: 122) PBGD mPBGD F TTGTACCCTGGCATACAGTTTGA (SEQ ID NO: 123)mPBGD R GTTCCCACGGCACTTTTC (SEQ ID NO: 124) HU: HUMAN HPRT: HYPOXANTHINEPHOSPHORIBOSYLTRANSFERASE MU: MOUSE PBGD: PORPHOBILINOGEN DEAMINASE

TABLE 3a PDE Cell lines Isotype A549 U937 EOL-1 Jurkat Hut-78 HL-60Daudi NCI-H292 PDE3A ++ − − − − − − − PDE3B ++ ++ ++ ++ ++ ++ − ++ PDE7A++ ++ ++ ++ ++ + + ++ PDE7B − − − − +/− − − − PDE4A +/− + +++ +++ +++++ + + PDE4B +++ ++ ++ − +++ − + ++ PDE4C − − − − + − − − PDE4D ++ − + −++ − − +

TABLE 3b PDE Primary cells Tissue Isotype NHBE BSMC Macro. Lung BrainPDE3A − + − + ++ PDE3B − ++ − +++ ++ PDE7A + + + ++ ++ PDE7B +/− − − − −PDE4A ++ − ++ ++ ++ PDE4B +/− ++ + +++ +++ PDE4C +/− − − +/− +/−PDE4D + + + ++ +

TABLE 4a Human PDE antisenses primary screening PDE Cell AntisenseAntisense % of mRNA Isoform Type Dose ID Inhibition PDE3A A549 1 ugTOP1312  6% TOP1308 54% PDE4A PBMC 2 uM TOP1406 −15%   TOP1411 43% PDE4BPBMC 5 uM TOP1432 14% TOP1437 53% PDE4D PBMC 2 uM TOP1495  3% TOP149856% PDE7A PBMC 5 uM TOP1711  5% TOP1706 40% PDE3B PBMC 0.1 uM TOP1351 4% TOP1357 75%

TABLE 4b Human PDE single antisenses with multiple gene knock downeffects Targeted PDE Antisense Antisense Cell % of PDE mRNA InhibitionIsoform ID Dose Type 3A 3B 4A 4B 4D 7A PDE3A TOP1312 1 ug A549 12% 16%18% — 16%  6% TOP1308 60% 53% 63% — 26% 44% PDE3B TOP1352 0.05 uM PBMCs— −51%   — — — — TOP1357 43% 73% 24% 28% 38% PDE4A TOP1404 2 uM PBMCs —−30%    1% −10%    −4%   −18%   TOP1413 56% 76% 82% 63% 25% PDE4BTOP1432 5 uM PBMCs —  −7%    5% 15% 15% −14%   TOP1437 42% 33% 60% 53%27% PDE4D TOP1494 2 uM PBMCs — 12% 13% 26% 15% 12% TOP1490 77% 94% 79%78% 64% PDE7A TOP1711 5 uM PBMCs —  7% — —  −7%    5% TOP1706 38% 70%49% 58% 40%

TABLE 5 Mouse PDE antisenses in vivo primary screening Antisense TargetAntisense Sequence Accession SEQ ID Identification Gene (5′-3′) TargetSequence Number NOS TOP2406 PDE4A TGTGCTAAGAGGTCCTC GAGGACCTCTTAGCACABC027224 125/126 TOP2410 PDE4A AGACTCATCGTTGTACAT ATGTACAACGATGAGTCTBC027224 127/128 TOP2411 PDE4A ACCATGTCGATGACCATCTT AAGATGGTCATCGACATGGTBC027224 129/130 TOP2412 PDE4A ACCATAGTCTTCAGGTCAG CTGACCTGAAGACTATGGTBC027224 131/132 TOP2426 PDE4B CTAGTTCCTCCAGCGTCTCC GGAGACGCTGGAGGAACTAGBC023751 133/134 TOP2430 PDE4B CATCTCTGAGAGGTGTGTC GACACACCTCTCAGAGATGBC023751 135/136 TOP2433 PDE4B GACAGAGCGGTAGGTCTG CAGACCTACCGCTCTGTCBC023751 137/138 TOP2435 PDE4B CTGATTGGAGACTCCAGG CCTGGAGTCTCCAATCAGBC023751 139/140 TOP2437 PDE4B GATGGACAATGTAGTCAAT ATTGACTACATTGTCCATCBC023751 141/142 TOP2707 PDE7A TCCAGATCGTGAGTGGC GCCACTCACGATCTGGABC062909 143/144 TOP2711 PDE7A CTTGCTTAATTCCCAGTTC GAACTGGGAATTAAGCAAGBC062909 145/146 TOP2712 PDE7A GTCAGAACCAGTTCTTC GAAGAACTGGTTCTGACBC062909 147/148 TOP2713 PDE7A GGAGCAATCTTACAGCTTC GAAGCTGTAAGATTGCTCCBC062909 149/150

1. A pharmaceutical composition for treating disease associated with reduced cAMP levels, said composition comprising a pharmaceutically acceptable carrier and at least two antisense oligonucleotide compounds each being capable of inhibiting expression of a target PDE, each oligonucleotide being directed against at least a portion of a nucleic acid sequence encoding a different target PDE, each oligonucleotide compound being present at a concentration that exhibits less than 20% inhibition of its target PDE, the combination exhibiting more than 20% inhibition and at least doubling the inhibition of at least one target PDE.
 2. A composition as defined in claim 1, wherein the combination exhibits inhibition of more than one target PDE.
 3. A composition as defined in claim 1, wherein the combination exhibits inhibition of other inflammatory genes selected from the group consisting of other PDE isotypes; cytokines; chemokines; inflammatory mediators; interleukins; TNF-alpha, and matrix metalloproteinases.
 4. A pharmaceutical composition according to claim 1, wherein the oligonucleotide compounds are directed against a nucleic acid coding for a PDE selected from the group consisting of PDE3A, PDE3B, PDE4A, PDE4B, PDE4D, PDE7A, and PDE7B.
 5. A pharmaceutical composition according to claim 1, comprising an oligonucleotide compound directed against a nucleic acid coding for PDE7A selected from the group consisting of SEQ. ID NO.: 1; SEQ. ID NO.: 2; SEQ. ID NO.: 3; SEQ. ID NO.: 4; SEQ. ID NO.: 5; SEQ. ID NO.: 6; SEQ. ID NO.: 7; SEQ. ID NO.: 8; SEQ. ID NO.: 9, and an oligonucleotide compound directed against a nucleic acid coding for PDE4B selected from the group consisting of SEQ. ID NO.: 27; SEQ. ID NO.: 28; SEQ. ID NO.: 29; SEQ. ID NO.: 30; SEQ. ID NO.: 31; and SEQ. ID NO.:
 32. 6. A pharmaceutical composition according to claim 1, comprising an oligonucleotide compound directed against a nucleic acid coding for PDE7A selected from the group consisting of SEQ. ID NO.: 1; SEQ. ID NO.: 2; SEQ. ID NO.: 3; SEQ. ID NO.: 4; SEQ. ID NO.: 5; SEQ. ID NO.: 6; SEQ. ID NO.: 7; SEQ. ID NO.: 8; SEQ. ID NO.: 9, and an oligonucleotide compound directed against a nucleic acid coding for PDE4D selected from the group consisting of SEQ. ID NO.: 33; SEQ. ID NO.: 34; SEQ. ID NO.: 35; and SEQ. ID NO.:
 36. 7. A method of treating a disease associated with reduced cAMP, the method comprising administering to a subject at least two antisense oligonucleotide compounds, each oligonucleotide being directed against at least a portion of a nucleic acid sequence encoding a different target PDE, wherein each oligonucleotide compound is capable of inhibiting expression of a target PDE, each oligonucleotide compound being present at a concentration exhibiting less than 20% inhibition of its target PDE, the combination exhibiting more than 20% inhibition and at least doubling the inhibition of at least one target PDE.
 8. A method as defined in claim 7, wherein the disease is a PDE-related disease.
 9. A method of treating a disease associated with reduced cAMP, the method comprising administering to a subject the pharmaceutical composition of claim
 1. 10. A formulation, comprising the composition of claim 1, selected from the group consisting of systemic and topical formulations.
 11. The formulation of claim 10, selected from the group consisting of oral, intrabuccal, intrapulmonary, rectal, intrauterine, intratumor, intracranial, nasal, intramuscular, subcutaneous, intravascular, intrathecal, inhalable, transdermal, intradermal, intracavitary, implantable, iontophoretic, ocular, vaginal, intraarticular, otical, intravenous, intramuscular, intraglandular, intraorgan, intralymphatic, implantable, slow release, and enteric coating formulations.
 12. The formulation of claim 11, which is an oral formulation, wherein the carrier is selected from the group consisting of solid and liquid carriers.
 13. An in vivo method of delivering a pharmaceutical composition to a target polynucleotide, comprising administering to a subject the composition of claim 1, said composition comprising an amount of carrier and oligonucleotide compound which permits the composition to reach the target PDE nucleic acid.
 14. A composition as defined in claim 1, wherein the oligonucleotide compounds are selected from an antisense oligonucleotide, a modified antisense oligonucleotide, an siRNA, and a ribozyme.
 15. A formulation as defined in claim 10, for topical administration.
 16. An article of manufacture comprising packaging material contained within which is an antisense oligonucleotide composition that is therapeutically effective to treat a disease associated with reduced cAMP in a subject, said composition comprising a pharmaceutically acceptable carrier and an antisense oligonucleotide compound that is effective to inhibit a PDE gene, said oligonucleotide compound being at least 50% complementary to the gene, said packaging material comprising a label which indicates that the composition is useful to treat said disease.
 17. An article of manufacture comprising packaging material contained within which is the composition of claim 1 that is therapeutically effective to a disease associated with reduced cAMP in a subject, the packaging material of the article comprising a label indicating that the composition can be used to treat said disease.
 18. An article of manufacture as defined in claim 17, wherein said label indicates that the composition is used to treat PDE-related disease.
 19. The composition as defined in claim 1, wherein at least a portion of the oligonucleotides hybridizes with RNA to form an oligonucleotide-RNA duplex
 20. The composition as defined in claim 1, wherein one of the at least two antisense oligonucleotides is directed against PDE7A.
 21. The composition as defined in claim 22, wherein another one of the at least two antisense oligonucleotides is directed against PDE4B.
 22. The composition as defined in claim 23, wherein the antisense oligonucleotide compound directed against PDE4B additionally inhibit expression of PDE4D.
 23. The composition as defined in claim 24, wherein the antisense oligonucleotide compound directed against PDE4B has no homology to the nucleotide sequence encoding the PDE4D gene.
 24. The composition as defined in claim 22, wherein another one of the at least two antisense oligonucleotides is directed against PDE4D.
 25. The composition as defined in claim 23, wherein the antisense oligonucleotide compound directed against PDE4D additionally inhibit expression of PDE4B.
 26. The composition as defined in claim 24, wherein the antisense oligonucleotide compound directed against PDE4D has no homology to the nucleotide sequence enclosing the PDE4B gene. 